Energy transduction by bioelectrochemical systems

Energy transduction by bioelectrochemical systems

193 Bioelectrochemistry and Bioenergetics, 11 (1983) 193-230 A section of J. Electroanal. Chem., and constituting Vol. 156 (1983) Elsevier Sequoia S...

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193

Bioelectrochemistry and Bioenergetics, 11 (1983) 193-230 A section of J. Electroanal. Chem., and constituting Vol. 156 (1983) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

Review article 599w ENERGY TRANSDUCTION

BY B I O E L E C T R O C H E M I C A L

SYSTEMS *

M. LOSADA, M. HERVAS, M.A. DE LA ROSA and F.F. DE LA ROSA

Departamento de Bioquimica, Facultad de Biologia, Universidad de Sevilla y C.S.L C., Sevilla (Spain) (Manuscript received July 22th 1983)

SUMMARY Electronic energy seems to be the obligatory link between the different forms of energy (light, redox, acid-base, phosphate-bond) transduced by the bioelectrochemical systems. These energy-transducing systems can operate, according to their nature and depending on their energization state, either at two alternate midpoint redox potentials (Ud and Ud*), or at two p K , ' s (pK~ and pK*), or at two phosphate transfer potentials (PTP and PTP*). The key point in energy coupling between any two of these biochemical systems lies apparently in the fact that both of them share a c o m m o n intermediate, which cyclically participates in the overall process by alternating between its electronically energized state and its unenergized basal state. Electronic energization of the coupling intermediate may proceed in one or two steps and can oscillate between approximately 0.2 and 1 eV molecule -1, i.e., between 20 and t00 kJ m o l e - 1.

I N T R O D U C T I O N **

Life on our planet depends in an admirable manner and almost absolutely on the radiant energy of sunlight that unceasingly reaches the biosphere in huge amounts (1017 W = 3 x 10 24 J year-t). As a matter of fact bioenergetics starts from water with the conversion of sunlight energy into electronic energy and subsequently into redox energy by the reaction centers chlorophyll a of the photosynthetic apparatus. The photosynthetically generated redox energy is further partly transformed, again through electronic energy, firstly into acid-base energy and secondly into phosphate-bond energy by the relevant transducing systems to be discussed below. This phosphate-bond energy is then used back, when required, to increase the redox energy level previously achieved by the light systems to a higher level (Fig. 1), capable by itself of directly reducing the oxidized primordial bioelements, namely, carbon dioxide, nitrate, dinitrogen and sulfate [1-7]. * Opening lecture at the 7th International Symposium on Bioelectrochemistry, Stuttgart, F.R.G., July 18-22, 1983. * * For easier comparison, all diagrams are presented in such a way that energy values are given upwards and that the symbols H and L before the symbol of a certain quantity stand for High and Low, respectively.

194 Solar energy transduction by the sequential action of all these systems leads finally to its storage as chemical redox energy in plant biomass (reduced carbon, nitrogen and sulfur), on one hand, and in molecular oxygen on the other (1014 W = 3 × 10 2~ J year -1 = 2 × 1014 kg biomass y e a r - l ) . The overall photosynthetic anabolic process necessitates the incessant p u m p i n g of an enormous n u m b e r of electrons (6 × 1032 e - s -1 = 3 × 10 21 C year -1) to a potential difference of about 1 V. The energized electrons, stored in biomass, return afterwards in a stepwise m a n n e r to molecular oxygen through the dissimilatory pathways of the primordial bioelements, in a first stage, and through the respiratory chain, in a second stage, thus closing the cycle and allowing the transformation of redox energy into phosp h a t e - b o n d energy directly, at the substrate level, and indirectly, through a c i d - b a s e energy, at the m e m b r a n e level (Fig. 2). In other words, not only electrons, but also protons and phosphate anions participate in astronomically high numbers in this most fundamental cycle of bioenergetics [2-5]. In contrast with the bacteriorhodopsin photosystem from h a l o b a c t e r i a - - w h i c h transduces light energy into electronic energy and subsequently into acid-base e n e r g y - - t h e chlorophyll photosystems are p e r se primary transducers of light energy into redox energy (Fig. 3). However, since the substrates that they eventually oxidize and reduce are, respectively, water (which acts as electron d o n o r in the intrathylakoid space) and the oxidized bioelements (which act as electron acceptors, A, in the extrathylakoid space), the chlorophyll photosystems also behave secondarily as transducers of light energy into a c i d - b a s e energy (Fig. 4), with the liberation on each side of the thylakoid m e m b r a n e of one proton and one hydroxyl ion per electron transferred, according to the following equations: light

2H20---, 4e-*+4H++O2 p*

A+4e-*+4H20

I.

~ AH4+4HO

+•

p*

e -roL-

txl e-

H+

Pi

e-~

Fig. 1. Sequential transduction of light energy into redox energy, acid-base energy, phosphate-bond energy and again redox energy during the photosynthetic process. Photons (hu) energize electrons (e ~ e- *); subsequently, electrons energize protons (H + ---,H + *); protons in turn energize phosphate (Pi --" - P*), and finally energy-rich phosphate groups further energize electrons (e * --, e- **). Electronic energy ( - ) seems to be the obligatory link between the different forms of energy transduced by these bioelectrochemical systems.

195

d

v

~p,~

h~

Fig. 2. The electron cycle of bioenergetics. Electrons from water are moved uphill to the potential level of the hydrogen electrode (e-*), at the expense of light energy (hv), during the light phase of photosynthesis, and further up to the potential level of the physiological electron acceptors (carbon, nitrogen and sulfur), at the expense of phosphate-bond energy ( - P*), during the dark phase of photosynthesis. Afterwards, the electrons from the reduced bioelements fall downhill to the potential level of redox coenzymes (e- *), during the dissimilatory processes proper to catabolism, and further down in cascade to molecular oxygen during the aerobic phase of respiration. Redox energy is eventually transduced into phosphate-bond energy ( - P*) through coupled phosphorylation at the substrate and membrane level during these dissimilatory and respiratory processes. The overall cycle involves the incessant turning over of about 1033 e - s 1, which rise and drop about 1 V between the redox level of the H 2 0 / O 2 pair and the redox level of the reduced/oxidized bioelements (A ~ A ) .

I t s h o u l d b e s t r e s s e d - - a s is c l e a r f r o m t h e a b o v e r e a c t i o n s - - t h a t p h o t o s y n t h e s i s i n s e n s u lato d o e s n o t c o m e to a n e n d w i t h t h e p h o t o l y s i s o f w a t e r i n t o h y d r o g e n , i.e., e l e c t r o n s a t t h e p o t e n t i a l level o f t h e h y d r o g e n e l e c t r o d e , a n d m o l e c u l a r o x y g e n . U s u a l l y [ 2 - 5 ] , t h e r e d u c i n g p o w e r s u p p l i e d b y t h e p h o t o d e c o m p o s i t i o n o f w a t e r is n o t a c c u m u l a t e d b y t h e g r e e n cell as m o l e c u l a r h y d r o g e n , b u t is r a t h e r t r a n s f e r r e d , with the aid of phosphate-bond energy when required, through suitable electron

h~~

e-~(H÷~)

w ~e-(H

+)

Fig. 3. Transduction of light energy (h~,) into acid-base energy (H + ---, H + *) by the bacteriorhodopsin system of halobacteria (left), and into redox energy (e ~ e - *) and acid-base energy (H + ~ H + *) by the reaction centers chlorophyll a of green plants (right). Electronic energy ( - ) is the obligatory link between the different forms of energy being transduced.

196

-¢ A+4H20

~

AH/. ÷ 4 H O I

_

Fig. 4. The photooxidation of water in the intrathylakoid space and the - P*-dependent reduction of the bioelements in the extrathylakoid space during the light and dark phases of photosynthesis. Eight light quanta ( h u ) are required for the uphill transfer of four electrons (e ) from water to the oxidized bioelements through the sequential action of the two photosystems (e *). The redox process is accompanied by the liberation of one proton in the intrathylakoid space and the fixation of one proton in the extrathylakoid space per each electron transferred.

carriers (ferredoxin and pyridine nucleotide) to the oxidized bioelements during the dark phase of photosynthesis and remains stored as such in the cell material (carbohydrates, hydrocarbons, lipids, proteins, nucleic acids, etc.). Actually [2-5], only in the case of nitrate is the photosynthetically generated reducing power energetic enough to promote the reduction of inorganic nitrogen from nitrate (oxidation state + 5) to ammonia (oxidation state - 3). By contrast, the reduction of molecular nitrogen (0) to ammonia ( - 3 ) , as well as the reduction of carbon dioxide (+ 4) to carbohydrate (0) and the reduction of sulfate ( + 6) to sulfide ( - 2), requires, in addition, adenosine triphosphate. This carrier of chemical energy is also formed during the light phase of photosynthesis, as mentioned above, by conversion of part of the photosynthetically generated redox energy into acid-base energy--which adds to that previously accumulated--and eventually into phosphate-bond energy through the consecutive action of the transducing cytochromes and the ATP synthetase, built in the thylakoid membrane, during the processes of cyclic and noncyclic phosphorylation. The following overall equations summarize the assimilatory reduction of the primordial oxidized bioelements: NO 3- + 8 e-* + 10 H 2 0 N2+6e

*+8H20

CO2+4e-*+4H20

~

12 - P*

~

3-P*

~

NH~- + 3 H 2 0

+

10 HO

2NH~-+8HO(CHzO)+HzO+4HO-

3-P*

S O 4 2 - + S e - * + 1 0 H 2 O --, H 2 S + 4 H z O + 1 0 H O -

197 ENERGY-TRANSDUCING REDOX, ACID-BASE AND PHOSPHATE-BONDSYSTEMS

It is our criterion [8-10] that the primary essential step in energy transduction (either light-redox, light-acid/base, redox-acid/base, redox-phosphate bond, or acid/base-phosphate bond) by bioelectrochemical systems is the generation of an electroniea[ly energized intermediate, i.e., an unstable atomic or molecular electronic configuration which tends to become stabilized by electron transition from a higher energy orbital to a lower energy orbital. Electronic energy seems thus to be the obligatory link between the different forms of energy transduced by the bioelectrochemical systems (Fig. 5). In order to introduce the subject, let us first discuss the redox systems and acid-base systems. We have proposed [2-5,8-11] that these energy-transducing systems operate on a basic common principle between two alternate midpoint redox potentials, Ud, or pKa's in carrying out their respective uphill (endergonic) or downhill (exergonic) electron or proton transfer between two potentials or pH's. In all cases, the corresponding change in redox potential or p K a is brought about by energization of one of the two forms of the pertinent couple. As is schematically shown in Fig. 6, energization (either photonic or chemical) of the reduced form (Red* versus Red) of the basal redox couple ( R e d * / O x versus R e d / O x ) determines an increase in its electron pressure, or in its dissociation constant for electrons, i.e., a decrease in its midpoint redox potential. On the other hand, energization of the oxidized form (Ox* versus Ox) of the basal redox couple ( R e d / O x * versus R e d / O x ) brings about a decrease in its electron pressure, i.e., an increase in its redox potential, or in its affinity for electrons. The two pairs of the energized reduced-form type systems ( R e d / O x and R e d * / O x ) share the same oxidized form and operate between the higher redox potential of the basal pair (HU~) and the lower redox potential of the energized pair (LUg*). The two pairs of the energized oxidized-form type systems ( R e d / O x and R e d / O x * ) share the same

LN IG TY E EH RG

I

,/"

AE C N D IEB R -G AS YE

]

I

energy

D O X ER NE E R G Y

",,,.

I

PHOS E P N H ER AG TE YB -OND I

Fig. 5. Energy transduction between the different forms of energy (light, redox, acid-base and phosphate-bond energy)by bioelectrochemicalsystems seems to involveelectronicenergy as the obligatory link.

198

,u7

Red~" Ox

aJ > aa

o~ >

Hu; uJ

r ........

Red /

,

Ox

Red/Ox

k ........

1 i

Red / Ox

energized reduced form

LU

Red/Ox ~ energized oxidized f o r m

Fig. 6. Energy-transducing redox systems of the energized reduced- (left) and oxidized-form (right) type. Upon electronic energization (either photonic or chemical) of the reduced form of the pair (Red ---,Red*), its midpoint redox potential decreases from its high basal value (HUd) to a lower value (LUd*), i.e., the energy level of the pair as related to its electron pressure increases (left). Similarly, upon electronic energization of the oxidized form of the pair (Ox ~ Ox*), its midpoint redox potential increases from its low basal value (LUd) to a higher value (HUd*), i.e., the energy level of the pair as related to its electron pressure decreases, and its electron affinity increases (right).

reduced form and operate between the lower redox potential of the basal pair (LUd) and the higher redox potential of the energized pair (HUd*). Both types of systems, however, transduce redox energy with the same net result. Ordinary redox pairs [12] modify their potential, U', in one or the other direction, with respect to their midpoint potential, Ud, when the concentration ratio between the oxidized and the reduced form of the pair moves away from its standard value of 1. Such changes follow the well-known Nernst equation, that for a redox couple involving n electrons is at 30 °C: U' = U~ + 0.06 log [Ox] n [Red]

(V)

According to this equation, a change in the [Ox]/[Red] ratio of 10 + 1 with respect to the standard value of 10 o brings about, respectively, a potential increase or decrease with respect to Ud of 60 mV in the one-electron couples, of 30 mV in the two-electron couples, and of 60/n mV in the n-electron couples. In general, to a [Ox]/[Red] ratio of 10 +p corresponds a redox potential U ' = Ud _+ (60p/n) (mV). For example, about +1 V for a 10 ±1~ ratio and n = 1, and about _+360 and +180 mV for a 10 -+6 ratio and n = 1 and 2, respectively. By contrast, energy-transducing redox systems modify their basal midpoint potential, in one or the other direction, by energization of either the reduced or the oxidized form of the couple rather than by changing the [Ox]/[Red] ratio. The energized form (Red* or Ox*) of the pertinent system is obviously unstable and tends spontaneously to transform itself into its corresponding unenergized one (Red or Ox). These transformations are consequently exergonic and determine a marked

199

deviation to the right of the equilibrium between the energized and the unenergized form, both in the case of the transducing systems of the energized reduced-form type: Red*~----~Red

(AG; < 0;

K'~q>

10 °)

and in the case of the transducing systems of the energized oxidized-form type:

ox*

ox

(aG < o; K;q > loo)

Since energization of either form of the basal redox pair is equivalent to an increase in its concentration of a certain number of times, as determined by its equilibrium constant, and since such an energization brings about a change in the midpoint potential of the pair with respect to the basal value, the relationship among the three quantities involved can be easily established. According to Ref. 4, the decrease in the midpoint redox potential, AU(~= U(~* - /_)~, brought about by energization of the reduced form of the basal couple in transducing systems of the energized reduced-form type, is given by the equation: AUg(. Red*-. Red) -.-

AG~ n.

0.06 n log K'q

(V)

Similarly, the increase in the midpoint redox potential, AU0 = U~*- U~, brought about by energization of the oxidized form of the basal couple in transducing systems of the energized oxidized-form type, is given by the equation:

aG(o~,_ox)-

AG~ = 0.06 log

,

,

K'q

(v)

In both cases, free-energy changes correspond to de-energization of the energized form (AG~ < 0; K~q>10 0) and are expressed in eV molecule 1. It can, thus, be estimated that energization by 60 meV molecule- 1 ( _~ 6 kJ m o l - 1 _ 1.4 kcal mol 1) of the reduced form of a one-electron transducing system--equivalent to a 10-fold increase of its concentration--determines a 60 mV decrease of the basal midpoint potential. For a two-electron transducing system, however, energization of the reduced form by 60 meV--also equivalent to a 10-fold increase of its concentration - - w o u l d bring about a decrease of only 30 mV in the midpoint potential of the basal redox couple. Analogous considerations can be applied to the transducing systems of the energized oxidized-form type, but taking into account that, in these cases, energization brings about an increase rather than a decrease of the basal midpoint potential. The great advantage of energy-transducing redox systems seems to be that, upon energization of either form of the basal couple, their midpoint potential changes enormously, in spite of keeping their [Ox]/[Red] ratio unaltered. For example, energization by I eV of the reduced or oxidized form of an one-electron system-such as reduced chlorophyll a or oxidized flavin--might produce a change in the basal

200

midpoint potential of 1000 mV, equivalent to a concentration increase of the corresponding form of about 101g-fold. Since the energy content of a high-energy phosphate bond in ATP is of the order of 33 kJ mo1-1--- 330 meV molecule -1, energization by it of the reduced or the oxidized form of a one-electron transducing system--equivalent to a concentration increase of the pertinent unenergized form of about 106-fold--would produce a _+330 mV change in the basal midpoint potential. If, however, the energy transducing redox system involves two electrons instead of one, energization by such an energy-rich phosphate bond of the pertinent reduced or TABLE 1

Midpoint potential of different types of energy-transducing redox systems in their basal and energized(*) states U~ p H 7 (V)

(a) Redox /phosphate - bond svstems Pyruvate/Acetate, CO 2 Pyruvate, C o A / A c e t y l - C o A * , C O 2

- 0.70 - 0.53*

a-Ketoglu./Succinate, CO 2 a-Ketoglu., C o A / S u c c i n i l - C o A * , C O 2

- 0.67 -0.49*

Acetaldehyde/Acetate Acetaldehyde, CoA/Acetyl-CoA*

- 0.60 - 0.41"

G3P/PGA G3P, P i / D P G A *

- 0.54 - 0.29*

HSO3/SO 2 H S O 3 , A M P / A M P - S O 4 ~-(*)

-0.48 - 0.06*

A z o f e r r e d o x i n *ea / A z o f e r r e d oxin ox Azoferredoxinre a / A z o f e r r e d o x i n ox

- 0.40 * - 0.29

(b) Redox/light systems C h l o r o p h y l l a P~'0o/Chlorophyll a P7+o0 C h l o r o p h y l l a PT00/Chlorophyll a PT+0O

-0.60* + 0.40

C h l o r o p h y l l a P6*80/Chlorophyll a P6~o C h l o r o p h y l l a P680/Chlorophyll a P6~o

0.0" + 1.0

(c) Redox / a c i d - base systems Ferrocyt. b - 5 6 3 / F e r r i c y t , b-563 Ferrocyt. b-563/Ferricyt, b-563"

- 0.18 0*

Ferrocyt. b - 5 5 9 / F e r r i c y t , b-559 Ferrocyt. b - 5 5 9 / F e r r i c y t , b-559"

+ 0.13 + 0.34*

Ferrocyt. b - 5 6 5 / F e r r i c y t , b-565 Ferrocyt. b-565/Ferricyt, b-565"

- 0.03 + 0.25*

Ferrocyt. a ~ / F e r r i c y t , a 3 Ferrocyt. a 3 / F e r r i c y t . a 3

+ 0.15" + 0.38

201

oxidized form--equivalent also to a concentration increase of about 106-fold--would determine a midpoint potential change of ± 330/2 = ± 165 mV. Table 1 assembles the midpoint redox potentials of the basal and energized couples of several transducing systems of the energized reduced- and oxidized-form types involved in carbon, sulfur and nitrogen metabolism, as well as in photosynthesis and respiration [2]. Although the coupling mechanism between the different kinds of energy-transducing systems will be discussed in detail later on, it is advisable to demonstrate at this point, as Fig. 7 schematically shows, how a redox system of the energized oxidized-form type would electronically couple with an energy-requiring or an energy-yielding reaction. Such an energy coupling would allow, at the expense of electronic energy, the uphill transfer of electrons from an appropriate donor pair to a suitable acceptor pair or vice versa.

100 (_)

e-= ~

Reduced form (%) 50,I

I

I

(-)

-\..<_o -~L..\

Red/Ox

LUo e~

Red/Ox -~,---

Lu;

Energyi

Energy *

HU~*

Red / O x :~ HU°~ e-

e- ~ R e d

/Ox

.

HU;* " ~ • ,

(+)

,

0

(+)

50 100 Oxidized form (%)

Fig. 7. Diagrammatic representation of the operating mechanism of a transducing redox system of the energized oxidized-form type coupled with either an energy-yielding reaction (left) or an energy-requiring reaction (right). The transducing system functions, depending on the energization state of the oxidized form of the pair, at two alternate midpoint redox potentials. U p o n electronic energization of the oxidized form of the basal pair (Ox ~ Ox*), its midpoint redox potential increases from a lower value (LUg) to a higher value (HU~*). In this way, the high-potential pair can accept isopotentially energy-poor electrons ( e ) from a donor, and the low-potential pair can donate, also isopotentially, energy-rich electrons ( e - *), to an acceptor. Consequently electrons can be driven up at the expense of the consumed energy (left). Similarly, upon electronic deenergization of the oxidized form of the energized p a i r ( O x * ~ Ox), its midpoint redox potential decreases from a higher value (HU~*) to a lower value (LUg). In this way, the low-potential pair can accept isopotentially energy-rich electrons ( e - *) from a donor, and the high-potential pair can donate, also isopotentially, energy-poor electrons ( e - ) to an acceptor. Consequently electrons can fall down liberating energy (right).

202

LpV

AH/AtJ

r- . . . . . . . I AH/A-

"1

L .......

"

:

Lpffa

AH/A-

energized acid f o r m

>~

L,J AH/A-

*

energized basic f o r m

Fig. 8. Energy-transducing acid-base systems of the energized acidic- (left) and basic-form (right) type. Upon electronic energization (either photonic or chemical) of the acidic form of the pair (AH ~ AH*), its pK,, decreases from its high basal value (HpKa) to a lower value (LpK*), i.e., the energy level of the pair as related to its proton concentration increases (left). Similarly, upon electronic energization of the basic form of the pair (A ~ A-*), its pK a increases from its low basal value (LpKa) to a higher value (HpK*), i.e., the energy level of the pair as related to its proton concentration decreases, and its proton affinity increases (right). As stated above, energy-transducing acid-base systems seem to operate on the same fundamental principle as energy-transducing redox systems. As is schematically shown in Fig. 8, energization (either photonic or chemical) of the acidic form (AH* v e r s u s A H ) of the basal a c i d - b a s e couple ( A H * / A - v e r s u s A H / A - ) determines an increase in its p r o t o n concentration, or in its dissociation constant for protons, i.e., a decrease in its p K a. On the other hand, energization of the basic form (A * v e r s u s A - ) of the basal a c i d - b a s e couple ( A H / A - * v e r s u s A H / A - ) brings about a decrease in its proton concentration, i.e., an increase in its p K a, or in its affinity for protons. The two pairs of the energized acidic-form type systems ( A H * / A - and A H / A - ) share the same basic form and operate between the higher p K a of the basal pair ( H p K a ) and the lower p K a of the energized pair (LpK*). The two pairs of the energized basic-form type systems ( A H / A - and A H / A - * ) share the same acidic form and operate between the lower pK~ of the basal pair (LpKa) and the higher p K a of the energized pair (HpK*). Both types of systems transduce, however, a c i d - b a s e energy with the same net result. Ordinary a c i d - b a s e [13] couples modify their pH, in one or the other direction, with respect to their pKa, when the concentration ratio between the basic and the acidic form of the pair moves away from its standard value of 1. Such changes follow the well-known H e n d e r s o n - H a s s e l b a l c h equation: [A-] p H = pK~ + log [ A H ] According to this equation a change in the [ A - ] / [ A H ] ratio of 10 +-1 with respect to the standard value of 10 ° brings about a p H increase or decrease of 1 p H unit with

203

respect to the pK~ value, so that, in general, to a [A ]/[AH] ratio of 10 +-p corresponds a pH = pK~ _+p (pH units). By contrast, energy-transducing acid-base systems modify their basal p K , in one or the other direction, by energization of either the acidic or the basic form of the couple rather than by changing the [A-]/[AH] ratio. The energized form (AH* or A - * ) of the pertinent system is obviously unstable and tends spontaneously to transform itself into its corresponding unenergized one (AH or A-). These transformations are consequently exergonic and determine a marked deviation to the right of the equilibrium between the energized and the unenergized form, both in the case of the transducing systems of the energized acidic-form type: AH*~AH

(AG0<0;

Keq>lO°)

and in the case of the transducing systems of the energized basic-form type: A *~---->A-

(AG 0 < 0 ;

Keq>lO°)

Since energization of either form of the basal acid-base pair is equivalent to an increase in its concentration of a certain number of times, as determined by its equilibrium constant, and since such an energization brings about a change in the pK~ of the pair with respect to the basal value, the relationship among these three quantities can again be easily calculated. Accordingly [4], the decrease in the pK a, ApKa = pK*a - pK~, brought about, at 30 °C, by energization of the acidic form of the basal couple in transducing systems of the energized acidic-form type is given by the equation: AGO APK~(AH*-AH)-- 0.0~ --

log

Keq

(pH units)

Similarly, the increase in the p K a, ApK u = pK* - pKa, brought about by energization of the basic form of the basal couple in transducing systems of the energized basic-form type is given by the equation: ApK~{A *-A,--

AGO 0.06 -- log

Keq

(pH units)

It can be calculated that energization by 60 meV molecule-~ (-- 6 kJ mol-1 __ 1.4 kcal mol-~) of the acidic form of an acid-base transducing system--equivalent to a 10-fold increase of its concentration--determines a decrease of one unit in the basal p K a. Analogous considerations can be applied to the transducing systems of the energized basic-form type, but taking into account that, in these cases, energization brings about an increase rather than a decrease of the basal pkg. The great advantage of energy-transducing acid-base systems appears again to be that, upon energization of either form of the basal couple, their pKa changes enormously, in spite of keeping their [A-]/[AH] ratio unaltered. For example, energization by 1 eV of the acidic or basic form of an acid-base system--such as protonated bacteriorhodopsin or unprotonated flavin--might produce, leaving aside

204

for the moment the electrical gradient (see below), a change in the basal p K , of about 18 pH units, equivalent to a concentration increase of the corresponding form of about 10t8-fold. Since the energy content of a high-energy bond in ATP is of the order of 330 meV molecule-1 (see above), energization by it of the acidic or basic form of an acid-base system could produce a change of about __+6 pH units in the basal p K a, equivalent to a concentration increase of the pertinent form of about one million-fold. Likewise, energization by 165 meV would result in a pK u change of about + 3 pH units. Acid-base energy-transducing systems would electronically couple with energyyielding or energy-requiring reactions in a manner similar to that of the corresponding redox systems (see Fig. 7), thus allowing proton transfer against an electrochemical gradient or vice versa. The coupling mechanism itself will be discussed later. It is, moreover, worth remembering that certain acid base pairs are neutral-anionic (e. g., carboxylic acids/carboxylate anions) whereas others are cationic-neutral (e.g., imino-cations/imino-neutral) and others anionic-anionic with a different charge degree (e.g., Hphosphate 2-/phosphate 3 ). It is our belief [11] that the same common principle which applies to energy-transducing redox and acid/base systems, i.e., that they can operate at two alternate redox midpoint potentials or pKa's, respectively, is also proper to phosphate-bond systems. In other words, as shown in Fig. 9, a conjugate phosphorylated/dephosphorylated pair can operate at two phosphate transfer potentials ( P T P ) , either high or low, depending on the energization state of the unphosphorylated or phosphorylated form of the pair. In the case of a system of the energized unphosphorylated-form type, the basal

HP T P ~

LPTP

_~*/R

I

I RP/R energzied phosphorylated form

it- ........

,'

RP/R

,,

R~R

I

HPrP c tJ

RP/R~

LPTP ~

energized

unphosphorylafed form Fig. 9. Energy-transducing phosphate-bond systems of the energized phosphorylated- (left) and unphosphorylated- (right) -form type. Upon electronic energization of the phosphorylated form of the pair (RP ~ RP*), its PTP increases from its low basal value (LPTP) to a higher value (HPTP*), i.e. the energy level of the pair as related to its phosphate transfer potential increases (left). Similarly, upon electronic energization of the unphosphorylated form of the pair (R ~ R*), its PTP decreases from its high basal value (HPTP) to a lower value (LPTP*), i.e., the energy level of the pair as related to its phosphate transfer potential decreases, and its affinity for inorganic phosphate increases (right).

205

pair ( R P / R ) would exhibit a high phosphate transfer potential since it could dissociate phosphate at high concentration, or rather transfer it as an energy-rich phosphate group, - P*, which is in fact (see also the last section) a phosphonium group (+PO32 )*, or a meta-phosphate monoanion (PO3)*, according to the equation: High PTP

RP

~

R + - P*

By contrast, the corresponding energized pair ( R P / R * ) would exhibit a lower phosphate transfer potential since it could dissociate phosphate at low concentration as plain inorganic phosphate, Pi, or HPO 4- : Low PTP*

RP

~

R* + Pi

A system of this type would, for example, be that constituted by the following pairs: acyl-phosphate/stabilized carboxylate anion, on one hand, and acyl-phosphate/energized carboxylate anion*, or rather acylium cation* (see below), on the other. Phosphate-bond systems of the energized phosphorylated-form type may similarly exist, at least theoretically, and operate at two phosphate potentials between the following two pairs: High PTP*

RP*

~

R + - P*

Low PTP

RP

~

R+P

i

In this respect, it is worth remembering that after H20 removal from the low P T P compound 2-phosphoglycerate by enolase--an intramolecular redox reaction--the resulting phosphoenolpyruvate (PEP) is an energy-rich phosphorylated compound of very high phosphate transfer potential. Actually, when PEP dissociates, both the resulting enolpyruvate and the phosphonium groups are electronically energized compounds (AG6 = 280 and 340 meV molecule 1, respectively). The phosphate-bond systems are more similar to the redox than to the acid-base systems, since they increase or decrease the potential of the phosphate group they transfer rather than concentrate or dilute it. For example, the energy-rich phosphate group, - P*, of ATP, the energy content of which is about 330 meV, is equivalent to inorganic phosphate, Pi, at a concentration about 106-fold higher, and in fact, when it is transferred to a hydroxyl ion, viz., hydrolyzed, it stabilizes itself by acquiring the electronic configuration of the latter. Inorganic phosphate belongs, in a certain way, to the acid-base systems of the energized acidic-form type, as reflected by the fact that its very high third p K a ( = 12) decreases about five units upon energization to an acyl-phosphate or anhydride configuration. The energized form of the phosphate-bond systems seems to be an electronically energized intermediate, which may correspond either to the energized reduced- or

206

oxidized-form of an energy-transducing redox system, or to the energized acidic- or basic-form of an energy-transducing acid-base system, thus allowing, as will be discussed in detail in this article, energy coupling between any of these two kinds of systems. The key point of energy coupling seems thus to be that any two transducing systems that can couple share one and the same intermediate, which can exist in either of two forms: electronically energized or unenergized. An example of such a common intermediate for the three pertinent types of systems might be, in the very relevant cases of an aldehyde (redox system), a carboxylic acid (acid-base system), and an acyl-phosphate (phosphate-bond system), the corresponding conjugate carboxylate anion, either stabilized by resonance or energized as an acylium cation, as suggested by the following equations: (1) Redox system: Low U~

R-CHO

¢

R-COO-+

2 e-*

High Ut~* .

R-CHO

~

(R-C=O)* + 2 e-

(2) Acid-base system: Low p K .

2 R-COOH

~

2 R-COO-+

2 H +(*)

High pK*

2 R-COOH

~

(R-C=O)* + R-COO-+

H20

(3) Phosphate.bond system." R-COOP R-COOP

HPTP

~

R-COO-+-

LPTP*

~

P*

( R - C = O ) * + Pi

It should be considered (see also the next and last sections) that complete energization of either a phosphate or a carboxylate group requires two energy-rich electrons or protons. Moreover, a carboxylic acid can dissociate a proton at two different pKa's, depending on the energization state of the resulting carboxylate anion: Low p K .

R-COOH

~

R-COO

+ H +(*)

High pK*

R-COOH

~

(R-COO-)* + H +

A semi-energized carboxylate anion would thus be equivalent, from an energetic

207 point of view, to the monomeric anhydride of a carboxylic acid, whereas an acylium cation would likewise be equivalent to a dimeric anhydride of a carboxylic acid, or to two semi-energized carboxylate anions and two low-energy protons, as formulated above and symbolized by the following disproportion reaction: 2 (R-COO)*

+ 2 H+~ R-COO

+ (R-C=O)** + H 2 0

as well as to one stabilized carboxylate anion and two high-energy protons, as indicated by the following equation: (R-C=O)** + H 2 0 ~ R - C O O

+ 2 H +(*)

Stabilization by resonance, as a consequence of its hydrolysis, of such a twice-energized acylium cation, which for the purpose has been exaggeratedly labelled with two asterisks, may involve an energy loss of about 50 kJ mol 1___500 meV molecule-1 (see below), and is equivalent to a concentration change of 10S-fold. To finish this section, it is important to emphasize that whereas redox and phosphate-bond systems can operate both in solution and in membraneous structures, acid-base systems must necessarily act--because of their own n a t u r e - - b o u n d to membranes forming vesicles, as has been cleverly and intuitively advanced by Mitchell [14,15]. COUPLING BETWEEN REDOX ENERGY AND PHOSPHATE-BONDENERGY One of the best known and most illustrative examples of a biochemical system that can transduce phosphate-bond energy into redox energy is that which catalyzes the ATP-dependent reduction by NAD(P)H of 3-phosphoglycerate (PGA), via 1,3 diphosphoglycerate (DPGA), to glyceraldehyde-3-phosphate (G3P). This system, proper to gluconeogenesis and to the reductive pentose cycle, also operates reversibly during glycolysis, yielding ATP from ADP and P~ in the oxidation by N A D + of G3P to PGA [2,4]. Figure 10 represents diagrammatically the mechanism of the uphill 2-electron transfer (AU~ = --0.22 V) from the potential level of the N A D ( P ) H / N A D P + couple (U~, pH 7, - 0 . 3 2 V) to the potential level of the G 3 P / P G A couple (U~, pH 7, - 0 . 5 4 V) at the expense of the T high-energy phosphate bond from ATP. The midpoint potential of the basal couple ( G 3 P / P G A ) increases by 0.25 V upon electronic energization by ATP of its oxidized form, i.e., upon phosphorylation, or rather phosphonilation (see below), of the carboxylate group of PGA to DPGA. The potential level of the energized couple, G3P, P J D P G A , becomes thus even more positive (U~, pH 7, - 0 . 2 9 V) than that of the pyridine nucleotide, and electrons can then flow almost isopotentially from the donor pair level to the energized acceptor pair level. Since the energy supplied by ATP in its hydrolysis to ADP and Pi is about 0.33 eV molecule -1, it can only increase the midpoint potential of the basal 2-electron acceptor pair in 0.165 V. The reaction catalyzed by 3-phosphoglycerate kinase is thus slightly endergonic (AG~ = + 2 X 0.085 = +0.17 eV molecule-I),

208 U'o.PH7(V) -0,6~p*

G3P/PGA

2e-~

-0,5f~P* - 0,4 / -0,3

NAO(P) H/NAO(P)+

G3P,F~/ DPGA*

2e-

Fig. 10. Diagrammatic representation of the ATP-dependent reduction by NAD(P)H of 3-phosphoglycerate (PGA) to glyceraldehyde-3-phosphate(G3P), via 1,3-diphosphoglycerate (DPGA*). Two electrons are transferred uphill from the potential level ( e ) of the NAD(P)H/NAD(P) + couple ( -0.32 V) to the potential level ( e - * ) of the G3P/PGA couple (-0.54 V) at the expense of the electronic energy ( - 0.33 eV) of the high-energy phosphate-bond ( - P*) from ATP, which concomitantly deenergizes itself

to inorganic phosphate (Pi). Energization by ATP of PGA to DPGA* (0.5 eV) implies a potential increase of 0.25 V from the value of the basal pair (G3P/PGA) to the value ( - 0.29 V) of the energized pair (G3P, P~/DPGA*).

whereas the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase is slightly exergonic ( A G ~ = - 2 ×0.03 = - 0 . 0 6 eV molecule-l). In summary, the overall process results as somewhat endergonic (AG6= +0.11 eV molecule-I), according to the following sequence of reactions: P G A + ATP ~ D P G A + A D P

(AG£ = +0.17 eV molecule 1)

D P G A + N A D ( P ) H ~ G3P + Pi + N A D ( P ) +

(AG6 = - 0 . 0 6 eV molecule 1)

The conversion of PGA to D P G A by A T P thus requires 0.50 eV in order to electronically energize the carboxylate anion, which, in its free form, is stabilized by resonance. However, although ATP is the eventual energy-rich compound which energizes P G A to DPGA, the true intermediate involved in the reductive reaction by N A D ( P ) H is in fact a thioester between the energized substrate and a thiol group of the enzyme-protein. D P G A thus behaves as a mixed acid anhydride which can either transfer its high-energy phosphonium group (+ PO 2 )* to an acceptor, like ADP, or its high-energy acylium group ( R - C = O ) * to an acceptor, like a sulfhydryl group in the active site of its specific enzyme. From the above considerations it can be concluded that glyceraldehyde-3-phosphate ( R - C H O ) can dissociate its two electrons at two potential levels, low Ud or high U~*, depending on the energization state of the resulting stabilized carboxylate

209

anion ( R - C O O ) or energized acylium cation (R-C=O)*, as shown by the following equations: Low U~

R-CHO

~

R-COO-+2e-*

R - C H O HighU'J ( R - C = O ) * + 2 e R-COO-+ 2 e-* ~ (R-C=O)* + 2 ewhich can also be formulated more exactly as follows: R-CHO + H20 ~ R-COO-+ 2 e-* + 3 H + R - C H O ~ (R-C=O)** + 2 e - + H + R - C O O - + 2 e * + 2 H + ~ (R-C=O)** + 2 e - + H20 where the twice-energized character of the acylium cation is again emphasized by a double labelling. Similarly, 1,3-diphosphoglycerate ( R - C O O P ) can dissociate its phosphate group at two potential levels, i.e., with high or low phosphate transfer potential, depending again on the energization state of the resulting stabilized carboxylate anion ( R - C O O ) or energized acylium cation (R-C=O)*, according to the following equations: High P T P

R-COOP

R-COOP R-CO0-+

~

R-CO0-+

Low PTP*

- P*

(R-C=O)* + Pi

- P* ~ ( R - C = O ) * + Pi

There is an obvious similarity between the dissociation of the acyl-phosphate, either into a stabilized carboxylate anion and an energized phosphonium group--which is in a certain manner a phosphonium c a t i o n - - o r into an energized acylium cation and a stabilized phosphate anion, since both high-energy groups are actually twice energized and energetically equivalent (see also the last section). In consequence, there exists a coupling between phosphate-bond energy and redox energy through a common intermediate, namely, an electronically energized acylium cation and its corresponding stabilized carboxylate anion, as shown diagrammatically in Fig. 11 and summarized by the following equations: - P* + R - C O O - ~

Pi + ( R - C = O ) *

2 e-+ (R-C=O)* ~ 2 e * + R-COO- P* + 2 e - ~

Pi + 2 e - *

It deserves to be noted that, upon its de-energization to inorganic phosphate, the phosphonium group accepts an oxide anion from the stabilized carboxylate anion,

210

~o c tJ

Fig. 11. Coupling between phosphate-bond energy and redox energy through electronic energy. A phosphonium group ( - P*) deenergizesitself to inorganic phosphate (Pi) with the concomitantenergization of a carboxylate anion (R-C00-) to an acylium cation (R-C=O)*. Subsequently, the resulting acylium cation deenergizesitself to the original carboxylateanion, w~-ththe concomitantenergizationof two electrons(2 e- ---,2 e- *). which becomes energized to an acylium cation. This reaction is accompanied by the fixation of a proton by the highest pK, group of the stabilized phosphate anion. The whole process is reversible, i.e., phosphorylation of the acylium cation and its further conversion to a phosphonium group involves removal of a proton and an oxide anion from inorganic phosphate. Likewise, the reduction of a carboxylate anion by two energy-rich electrons is concomitant with the removal from it, as water, of an oxide anion (see also the last section), whereas the reduction of an acylium cation requires merely the acceptance of two relatively energy-poor electrons as a hydride ion. There exists in chloroplasts a very interesting NADP+-dependent glyceraldehyde3-phosphate dehydrogenase which catalyzes the oxidation of this triose phosphate by N A D P + in the absence of inorganic phosphate, the redox reaction being thus uncoupled from phosphorylation [2,4]. The effective donor pair is therefore the very reducing G 3 P / P G A (U~ = - 0 . 5 4 V), the reaction being very exergonic and irreversible: G3P + N A D P + ~ PGA + N A D P H

(AG~ = - 0 . 4 4 eV molecule 1)

Thioesterification is, moreover, an intermediate step in several energy-transducing reactions taking place during the assimilatory and dissimilatory pathways of carbon, viz., the reductive carboxylation of succinate to a-ketoglutarate (U~, pH 7, - 0.67 V) and of acetate to pyruvate (Ud, pH 7, - 0 . 7 0 V) by reduced ferredoxin (U~, pH 7, - 0 . 4 2 V), proper to the reductive tricarboxylic acid cycle, and the corresponding oxidative decarboxylations by NAD+ during the Krebs cycle. Adenylation also occurs as the energization step in the assimilatory and dissimilatory pathways of sulfur, viz., the reduction of sulfate to sulfite (U~, pH 7, - 0 . 4 8 V) by NAD(P)H or the corresponding oxidation. All the energy-transducing redox systems involved in carbon and sulfur metabolism (see Table 1) are of the energized oxidized-form type, i.e., the midpoint potential of the basal pair increases about 0.2 V - - o r even 0.4 V in the case of sulfate activation--upon electronic energization by ATP of the oxidized form of the pair.

211 U'o , pH 7 (V) e-~--xN 2 H2 / N 2 ~ Mo Fere d / M o F e o x

Pi -0.4

-0.3

MoFered /MoFe%xe-,~- Feted / Feox

Fete d / F % x ~

e-

Fig. 12. Schematic representation of possible energy-transducing systems involved in the reduction of dinitrogen to ammonia by nitrogenase. Electrons would be driven up by the electronic energy supplied by the phosphonium groups ( - P*) of ATP, which would concomitantly deenergize themselves to inorganic phosphate (P~). Electrons would be energized in a first stage (e----, e *) by the Fe protein system (energized reduced-form type) and again in a second stage (e- * ---,e **), corresponding to the reduction level of N 2 to N z H 2, by the MoFe protein system (energized oxidized-form type). A n o t h e r interesting energy-coupled redox reaction, proper to inorganic nitrogen metabolism, is the reduction by ferredoxin of molecular nitrogen to ammonia, which requires high amounts of A T P [16]. Although its mechanism remains as yet unsolved, it is likely that it m a y include one or more steps in which p h o s p h a t e - b o n d energy is transduced into redox energy. In this respect, it is remarkable that the reverse oxidation reaction appears to be coupled to the synthesis of A T P [16]. It is still difficult to predict whether energy-transducing systems of either the oxidized- or the reduced-form type are involved in the process, but Z u m f t et al. (cf. Ref. 17) have already reported that azoferredoxin shifts its potential by A T P from - 0 . 2 9 V to - 0 . 4 0 V. Based on this finding, a hypothetical mechanism, which slightly differs from that proposed recently by Z u m f t [17], is diagrammatically represented in Fig. 12. COUPLING BETWEEN LIGHT ENERGY AND REDOX ENERGY Transduction of light energy into electronic energy and subsequently into redox energy takes place in the two photosystems of the photosynthetic apparatus and is catalyzed by reaction center chlorophyll a. U p o n absorption of one q u a n t u m of red light by the reduced form of the pigment in its g r o u n d state (chl), it becomes photoexcited by raising one electron to a higher energy orbital (chl*). Experimental data show that the energy difference between the pigment in its photoenergized and ground states is about 1 eV, equivalent in concentration to an increase of about 1018-fold. Taking into account the energy content of the p h o t o n absorbed ( = 1.8 eV) this value represents an efficiency of about 55 % in the conversion of light energy

212

into electronic energy. A very important property of reaction center chlorophyll a in its photoexcited state is that it can donate its energized electro~a to appropriate electron acceptors, thus becoming oxidized (chl +) and able to receive again an electron in the original orbital of lower energy from suitable electron donors [2-5]. The reaction center chlorophyll a is obviously the first and most significant energy-transducing photosystem in biology. It belongs to the energy-transducing redox photosystems of the energized reduced-form type and can, therefore, exist (Fig. 13) in two redox pairs which share the same oxidized form: the high potential or basal pair, HU~,in which the reduced form is unenergized and stable (chl/chl+), and the low potential or energized pair, LU'~', in which the reduced form is energized and unstable (chl*/chl+). The reaction center chlorophyll a can thus promote the light-driven uphill transfer of electrons from an electron-donor pair of high potential to an electron acceptor pair of low potential [2-5]. As mentioned in the introduction, green plants can split, at the expense of visible light, water into hydrogen and oxygen, that is to say, they are able to raise electrons from the potential level of the pair H 2 0 / O 2 (U~, pH 7, + 0.82 V) to the potential level of the hydrogen electrode (U~, pH 7, - 0 . 4 2 V) in order to use them, with the concourse of additional phosphate-bond energy when required, for the reduction of the oxidized primordial bioelements. This light-driven uphill transfer of electrons is accompanied by the liberation of protons inside the intrathylakoid space and by the liberation of hydroxide anions in the extrathylakoid space, i.e. by the additional generation at the expense of visible light of an electrochemical gradient of protons. There is general agreement that this photosynthetic non-cyclic flow of electrons requires the cooperation of two photosystems operating in series [1-7]. As shown schematically in Fig. 14, pigment P680 of photosystem II removes electrons from water as the initial electron donor at + 1.0 V and delivers them to an intermediate carrier at 0.0 V. Pigment P700 of photosystem I afterwards accepts these electrons

LUo'~

-- e-*

ho

~

.Uo

~ L ° w U0 "~ c h l ~ / c h l + e- ~

ho

chl/chl +

~ High U°

e-

e-

Fig. 13. Transduction of light energy into electronic energy and subsequently into redox energy by a chlorophyll a system. Upon absorption of a photon (h u), the reduced form of the pigment (chl) becomes electronically energized (chl*). The redox couple in its basal state (chlflchl +) can accept electrons ( e - ) at a high potential (HUd) from appropriate donors, whereas the redox couple in its excited state (chl*/chl +) can donate electrons ( e - *) at a lower potential (LUg*) to suitable acceptors. In this way, chlorophyll a can promote the light-driven uphill transfer of electrons.

213 U o , pH

(v) e- J ~ _ _ . p -x- / p + 700 700

-0.5-

~'--

hp

H

+ P700/ Poo ~

+

ferrocyt.~ / fe r r i c y t . b

~,...- H+- ferrocyt, b / ferric I

H+ ~, e - % ~/ #

P* /P+ 680 680

b~X-H +

~"

+0.5

+ 1.0

~ o e P8o/P68

Fig. 14. Energy-transducingsteps in photosynthetic non-cyclic electron flow. Pigment systems P68oand Pv00of photosystemsII and I belong, respectively,to the energized reduced-form type and transduce light energy into redox energy (see Fig. 13). Cytochromeb-559 belongs to the energized oxidized-formtype and seems to transduce redox energy between the reduced side of photosystem II and the oxidized side of photosystem I into acid-base energy (see Fig. 24).

from another intermediate carrier at + 0.4 V and donates them at - 0 . 6 V for the eventual reduction of ferredoxin ( - 0 . 4 2 V), the terminal electron acceptor in the photosynthetic transport chain. An important observation is that the overall potential span includes a short downhill stretch between the two photosystems of about 0.3 V that with a remarkable sense of biological efficiency might allow the transduction of this redox energy into acid-base energy by cytochrome b-559 (see p. 217). Although the reaction center chlorophyll a is physiologically embedded in the thylakoid membranes, it can also function similarly when dissolved in organic solvents or aqueous solutions of detergents, using, for example, phenylhydrazine or cysteine as electron donor and methyl viologen as electron acceptor [18]. Another photosystem which can transduce light energy into electronic energy and subsequently into redox energy, or vice versa, is that represented by flavins [2-4]. By contrast with the chlorophyll systems, flavins are energy-transducing redox photosystems of the energized oxidized-form type, i.e. they increase their midpoint redox potential upon energization of the oxidized form of the pair. As shown schematically in Fig. 15, the fundamental feature of the flavin photosystem is that the yellow oxidized form (FN) is the light-absorbing species. In its photoexcited unstable form (FN*) the corresponding redox pair (FN~-/FN *) exhibits a redox potential higher than that of the unenergized pair ( F N ' / F N ) and is therefore able to accept electrons with higher affinity at a lower energy level. In this way, flavins can

214 U'

e- ~ L O W U 0

FN-/FN

F N 2 / F N ~< ~ HighU°#-

e-

e-

Fig. 15. Transduction of light energy into electronic energy and subsequently into redox energy by a flavin system. U p o n absorption of a photon (h J,), the oxidized form of the pigment (FN) becomes electronically energized (FN*). The redox couple in its excited state ( F N ~ / F N *) can accept electrons ( e - ) at a high potential (HUd*) from appropriate donors, whereas the redox couple in its basal state ( F N ~ / F N ) , can donate electrons (e *) at a lower potential (LUd) to suitable acceptors. In this way, flavins can promote the light-driven uphill transfer of electrons.

promote the light-driven uphill transfer of electrons from appropriate electron donors, such as EDTA or semicarbazide, to suitable electron acceptors, such as protons, molecular oxygen, nitrate, molecular nitrogen, etc., thus being valuable converters of sunlight energy into redox energy [19,20]. Although chlorophyll a and flavins belong, respectively, to redox photosystems of the energized reduced- and oxidized-form type, both function as light-driven electron pumps with the same net result. In both systems, the intermediate which couples light energy with redox energy is the pigment itself, either in its reduced state (chlorophyll a) or in its oxidized state (flavins). As shown schematically in Fig. 16, the corresponding photoactive species of the reduced or oxidized pigment

~ " c h l "~'-

~ e -

~

F

N

~

~-

e-

Fig. 16. Coupling between light energy and redox energy through electronic energy by a chlorophyll a (chl) system (energized reduced-form type) and a r a v i n (FN) system (energized oxidized-form type). In the first case (left), a photon ( h u ) energizes the reduced form of chlorophyll a (chl ~ chl*), which is then able to donate its energized electron (e *) to an acceptor, and subsequently to accept, in its oxidized form, an energy-poor electron ( e ) from a donor, thus regenerating the original reduced form. In the second case (right), a photon energizes the oxidized form of a r a v i n (FN ~ FN*), which is then able to accept an energy-poor electron ( e ) from a donor and subsequently to donate, in its reduced form, an energy-rich electron ( e - *) to an acceptor, thus regenerating the original oxidized form.

215

participates cyclically in the energy-transducing process by alternating between its electronically energized state and its unenergized basal state.

C O U P L I N G BETWEEN L I G H T E N E R G Y A N D A C I D - B A S E E N E R G Y

As mentioned in the Introduction, the primary transducing step in Halobacterium halobium photosynthesis differs essentially from that in green-plant photosynthesis in not involving light-driven electron transfer, but consists basically of the conversion by bacteriorhodopsin of light energy into electronic energy and subsequently into acid-base energy. Bacteriorhodopsin [21] is a purple pigment related to rhodopsin, the visual pigment of the mammalian retina; its prosthetic group is retinal (vitamin A) linked to a lysine residue by a Schiff's base of very high pK a. Upon illumination, under anaerobic conditions, the purple membrane translocates protons from inside the cell to the outside medium forming an electrochemical proton gradient across the membrane that can be used eventually to drive either ATP synthesis by a protontranslocating ATPase or the active uptake of cations such as potassium (see p. 227). Bacteriorhodopsin thus seems to belong to the energy-transducing acid-base photosystems of the energized acidic-form type that decrease their pK, upon energization [4,8-11]. As schematically shown in Fig. 17, upon absorption of one quantum of light at 560 nm by the protonated form of the pigment in its ground state (PH÷), it becomes photoexcited (PH ÷*). In its electronically energized state, acidic bacteriorhodopsin can dissociate a proton at a lower pKa, becoming eventually unprotonated in its original basic form (P). In other words, considering the

p!

LpKa*

H+ *

__

Low pKa"~

pH + -X-/p hp

pH+/p

High

p/f~

H+

H+ ~

H+

Fig. 17. Transduction of light energy into electronic energy and subsequently into acid-base energy by a photosystem of the energized acidic-form type. U p o n absorption of a photon (h ~,), the acidic form of the pigment (PH +) becomes electronically energized (PH+*). The acid-base couple in its basal state ( P H ÷ / P ) can accept protons (H ÷) at a high pK,, (HpKo), whereas the acid-base couple in its excited state (PH ÷ * / P ) can dissociate protons (H ÷ *) at a lower pK,, (LpK*). In this way, a transducing-pigment, such as bacteriorhodopsin or stentorin, could promote the light-driven uphill transfer of protons against an electrochemical gradient.

216

overall process, bacteriorhodopsin can exist in two acid/base pairs, which share the same basic form: the basal pair of high pK~ (HpKa), in which the protonated form is unenergized and stable (PH+/P), and the energized pair of low p K , (LpK*), in which the reduced form is electronically energized and unstable (PH +*/P). Purple membrane bacteriorhodopsin can thus promote the light-driven uphill transfer of protons from the inside medium of higher pH to the outside medium of lower pH, establishing an electrochemical proton gradient of about 200 to 300 mV. There is another very interesting pigment, the receptor of the photophobic blue-green ciliate Stentor, or stentorin, the chromophore of which has been identified as hypericin [22]. Irradiation of whole living Stentor in dilute buffer solutions induces a decrease in the pH of the medium, as a consequence of the lowering of the p K a from 7.0 to 3.5 of one or more hydroxyl groups of the excited chromophore. Lowering of p K a in the excited state thus seems a crucial characteristic of the energy-transducing biological photosystems of the energized acidic-form type. The phenomenon is also common to other more simple compounds, such as the phenol pyridoxamine [23], the pKa of which is shifted by 6.9 units from a value of 3.4 in the ground state to a value of - 3 . 5 in the photoexcited state. Flavins are remarkable pigments for they can transduce light energy not only into redox energy, but also into acid-base energy. In contrast with the acid-base photosystems described above, flavins are photosystems of the energized basic-form type, i.e., they increase their p K a upon energization of the basic form of the pair. As shown schematically in Fig. 18, the basic form in its fundamental unenergized state (FN) presents a very low affinity for protons, the pKa of the corresponding acid-base pair ( F N H + / F N ) being about 0. However, upon the absorption of one quantum of blue light by the basic form, it becomes electronically energized (FN*) and its affinity for protons increases enormously, the pK* of the corresponding

p~ Lp/'(d

H+ ~

Low

p Kd

+

FNH/FN

FNH+/FN * ~, H i g h p " ~ "x- H+

hi)

H+ *

H+

Fig, 18. Transduction of light energy into electronic energy and subsequently into acid-base energy by a photosystem of the energized basic-form type, such as a flavin. U p o n absorption of a photon (hv), the basic form of the pigment (FN) becomes electronically energized (FN*). The acid-base couple in its excited state ( F N H + / F N *) can accept protons (H +) at a high p K a (HpK*), whereas the acid-base couple in its basal state ( F N H ÷ / F N ) can dissociate protons (H + *) at a lower p K a (LpK~). In this way, flavins might promote the light-driven uphill transfer of protons against an electrochemical gradient.

217

hg~ PH+~ ~H+~ h~~ FN~ H+~ L,J

pH+~H + ~FN~H +

Fig. 19. Coupling between light-energy and acid-base energy through electronic energy by a pigment photosystem of either the energized acidic- (left) or basic-form (right) type. In the first case, a photon (hu) energizes the acidic form of a pigment, such as bacteriorhodopsin (PH+~ PH +*), which is then able to dissociate its energized proton (H + *) and subsequently to accept, in its basic form, an energy-poor proton (H+), thus regenerating the original acidic form (left). In the second case, a photon energizes the basic form of a pigment, such as ravin (FN ---,FN*), which is then able to accept an energy-poor proton (H +) and subsequently to dissociate, in its acidic form, an energy-rich proton (H + *), thus regenerating the original basic form (right).

a c i d - b a s e pair ( F N H + / F N *) being about 5 [24]. It is therefore plausible that flavins - - w h e n properly oriented in m e m b r a n e s - - c a n p r o m o t e the light-driven uphill transfer of protons and create a transmembrane proton gradient [4,25], as has been shown to be the case with bacteriorhodopsin or stentorin. In other words, although these latter pigments and flavins belong, respectively, to a c i d - b a s e photosystems of the energized acidic- and basic-form type, both can function as light-driven p r o t o n p u m p s with the same net result. In both types of systems the intermediate which couples light energy with a c i d - b a s e energy is the pigment itself, either in its acidic f o r m (bacteriorhodopsin, stentorin) or in its basic form (flavins). As shown schematically in Fig. 19, the corresponding photoactive species of the protonated or unprotonated pigment participates cyclically in the energy-transducing process by alternating between its electronically energized state and its unenergized basal state. COUPLING BETWEEN REDOX ENERGY AND ACID-BASE ENERGY The major working hypothesis for interpreting the mechanism of A T P synthesis coupled to electron transport in bacterial, mitochondrial or thylakoid membranes [26] is the chemiosmotic theory of Mitchell, which postulates that respiratory or photosynthetic electron transport is compulsorily coupled to p r o t o n transport in such a way that for each electron traversing an energy-coupling site a p r o t o n is translocated across the respective membrane, either from the bacterial cytosol or the mitochondrial matrix to the outside, or from the chloroplast stroma into the inner thylakoid space. The pertinent p r o t o n translocation results in all cases in the establishment of an electrochemical gradient across the corresponding m e m b r a n e (positive outside or inside, respectively). This proton-motive force, as it is also called, is c o m p o s e d of a proton concentration gradient and a m e m b r a n e potential (or electrical gradient) and constitutes the high-energy intermediate state that drives

218 ATP synthesis in oxidative and photosynthetic phosphorylation, when eventually the protons flow back via the FoF~-ATPase, or ATP synthetase, an otherwise remarkable transducing enzyme complex that is also embedded in the respiratory or photosynthetic membranes with its knob towards the inner or outer space, respectively (see p. 227). Although the chemiosmotic hypothesis accounts for many observations on energy transduction in bacteria, mitochondria and chloroplasts, there are still aspects of it that are little understood or are in contradiction with current evidence. Among these is the mechanism, in molecular terms, by which redox energy and acid-base energy, as well as acid-base energy and phosphate-bond energy, are coupled. This aspect of energy-transduction is precisely one of the main topics with which we will be concerned in the present article. First of all it should be pointed out that, according to our interpretation [2,4,9,10], several respiratory and photosynthetic cytochromes are to be considered energy-transducing redox systems of the energized oxidized- or reduced-form type, viz., redox systems which increase or decrease their midpoint potential upon energization of the oxidized or reduced form of the respective basal pair (see Table 1). As a matter of fact, Wilson and Dutton [27] and Chance et al. [28] made in 1970 the fundamental discovery that the midpoint potential of cytochrome b-565 from rat liver and pigeon heart mitochondria increases by about 250 mV upon energization by ATP. The reversible phosphate potential dependence of the midpoint redox potential of cytochrome b-565 seems to be a general phenomenon, and we ourselves have observed it in yeast mitochondria [29]. As shown in Fig. 20, yeast mitochondria exhibit most of their cytochrome b-564 in an unstable high-potential form (U~*, p H 7.2, 180 mV) when incubated in the presence of 10 m M ATP, whereas in the presence of 10 m M ADP, Pi, practically all of it is present in a stable low-potential form (U~, p H 7.2, 60 mV). ATP seems to exert its energization effect through the F0F~-ATPase, since no shift to the high-potential form is observed in the presence of oligomycin. On the other hand, addition of protonophoric agents, like FCCP, or structural alteration by disturbing agents, like sonication, detergents, or mild heating, causes the de-energization of the high-potential form and the irreversible displacement of the equilibrium towards the low-potential form. Another very important and remarkable observation made by Wilson and Dutton [30] is that the midpoint redox potential of the cytochrome a 3 component of mitochondrial cytochrome c oxidase decreases upon energization instead of increasing. Moreover, it seems to be well established at the present time that redox-linked vectorial proton translocation is one of the intrinsic catalytic properties of the bc~and aa3-type mitochondrial complexes [31-33]. In contrast with mitochondrial transducing-cytochromes, the function of the b-type photosynthetic cytochromes, although widely and intensely investigated, has not yet been sufficiently clarified [9,10,34-37]. In fresh chloroplasts (cf. Refs. 9 and 10), cytochrome b-559 occurs in two forms with identical light absorption spectra but with two different midpoint redox potentials: an unstable, energized high-potential form (U~*, p H 7, 340 mV), reducible by hydroquinone, and a stable, unenergized

219

No addition

ATP:10 mM U'oCmV)

ADPPi , : 10 rnM

/J. (my)

i

U'o(mV)

22C

./,

220t 180

HU;:.~ ¶8o

HU~8o -~'" I

,.-Y

Io+(°~-]-

I

tR+,] +,1

log[Redl+ ~ 1I

~ooJr

- ioof

60t 100

LU;o2/

|

,_,

tog [ox] /S O o

LU' /,....../o n=l %.0 060

/ 0 0=1 -60

o/

HU? 30 --~o "-~

HU~ 90 LU'o 10

Hu'o*

o

LU'°

100

Fig. 20. Yeast mitochondria cytochrome b-564 exhibits an unstable high-potential form (HU~*) and a stable low-potential form (LUg). Upon energization of mitochondrial preparations by ATP (left), most of the cytochrome (90 %) occurs in its high-potential, unstable form. Conversely, upon deenergetization by ADP, Pi (right), practically all the cytochrome (100 %) occurs in its low-potential, stable form (cf. Ref. 29).

low-potential form (U~, pH 7, 135 mV), reducible by dithionite. On average, fresh chloroplasts contain about 2/3 of their cytochrome b-559 in its high-potential form and about 1/3 in its low-potential form (Fig. 21). Both cytochrome b-559 potential forms are interconvertible, the low-potential form being pH-dependent with a pKo

Heated ¢hbroptasts

Fresh ch[oroptasts

I

i

AA=2xlO 3

A ,4= 2x10 -3

b-559HP+L P ./O....O

559Lp

.:."of ° ~ o "e.+ o"'/ X ""

•""/b-559

/

o "$

560 x (.r.)

\

/

~," "~')° i ' " Lpi " ,,,,~!.... 550

/\

570

'

~;o

",, s; o

STO

x (.m)

Fig. 21. Spinach chloroplasts present about 2 / 3 of their cytochrome b-559 in a high-potential (HP), unstable form, and about 1 / 3 in a low-potential (LP), stable form (left). Upon deenergization of chloroplast preparations by mild heating, practically all the cytochrome occurs in its low-potential, stable form (right). Both forms exhibit one and the same absorption spectrum (cf. Ref. 9).

220

ferricyt.b

I H+-ferrocyt. b ~

+ e-*+ H+ I

I ferrocyt.b ~ferricyt. b + e- ~

I

.60

608 0 . -- LL/o'



-%,,,

• -,.--.~.~ - - ~

e lOO-

m--

-80 2. E

.100-~ o

1

12o.

-120 ~tu

140-

340

LUo-

plus CCCP

minus CCCP

HUo'~- •





-140

1

Aq--

340 ~

_[-360 pH

9

8

7

6

pH

8

;

Fig. 22. Spinach chloroplast cytochrome b-559 exhibits a high-potential, unstable form (HUd*), that is pH-independent, and a low-potential, stable form (LUd), that is pH-dependent, with a p K , of 7.6 and a slope of about 60 m V per unit of pH (left). In the presence of the protonophoric agent C C C P ( 3 3 / * M ) , not only is the high-potential form converted into the low-potential one, but the latter becomes pH-independent (right). Each of the three redox reactions which may be involved are written close to their respective graphs (cf. Refs. 9 and 38).

of 7.6, whereas the high-potential form is pH-independent. Besides, we have now found [38] that in the presence of CCCP, a protonophoric uncoupler that converts the high-potential form into the low-potential one (see above), the low-potential form also becomes pH-independent (Fig. 22). In contrast, ammonia, which is likewise a potent uncoupler of photophosphorylation, does not affect the redox behavior of cytochrome b-559, its uncoupling action being more likely to be on either the proton gradient or the FoF1-ATPase itself. Although interpretation of all these facts has been a difficult task, it has ultimately shed new light on the coupling mechanism betwen redox energy and acid-base energy catalyzed by the mitochondrial and chloroplast complexes connected with the energy-conserving sites. In particular, the findings that the highpotential form of cytochrome b-559 is pH-independent and easily convertible in the low-potential form, whereas the latter is stable and pH-dependent with a slope of about 60 mV per unit of pH and a pK a of 7.6, becoming pH-independent in the presence of CCCP, have led to the following relevant conclusions, summarized by the equations collected in Fig. 22: (1) Reduction of the unenergized oxidized form of cytochrome b-559 (ferricyt. b) is forced to occur at low potential (LUg) and determines the appearance of a high pK a group in the corresponding reduced form, which in consequence becomes protonated at pH below such pK a. (2) Oxidation of the reduced and protonated cytochrome (H+-ferrocyt. b) at

221

high potential (HUo*) leads compulsorily to the formation of the energized oxidized form and determines the disappearance of the high pK~ group inherent to the reduced form, as well as the simultaneous reappearance of the high pK* group proper to the oxidized form in its energized state, which consequently becomes protonated (ferricyt. b*-H+). When this form dissociates its proton at low pK~,, it again becomes stabilized (ferricyt. b), closing the cycle. Alternatively, the energized oxidized form may dissociate its proton at high pK* (not shown), in which case it does not become stabilized and can use its energy for some other purposes. (3) Oxidation-reduction of cytochrome b-559 at high potential implies proton translocation from the high pK~ group proper to the reduced form to the high pK* group proper to the oxidized form in its energized state. (4) Protonation of the unenergized oxidized form is forced to occur at low pK~ and necessarily implies its electronic energization. (5) CCCP not only converts the high-potential form into the low-potential one by proton removal, but also impedes protonation of the reduced form (ferrocyt. b). This

Low ~ e- ~

@@@

A ferrocyt.b

ferricyt, b

I

H+

_L

HighUo~

H+ - ferrocyt, b

(~ I

H÷ ferricyt.~ ~

H+

Fig. 23. Chloroplast cytochrome b-559 is apparently both a transducing redox system that operates at two alternate midpoint redox potentials (LU~ and HU~*), and a transducing acid-base system that operates at two alternate pKa's (LpK a and HpK*). Both systems share a common intermediate, which can exist in either its unenergized form (ferricyt. b) or its electronically energized one (ferricyt. b*). The unenergized oxidized form can he reversibly converted into the energized oxidized one by either of two ways: (1) Reduction at low potential ( e - * ) - - c o n c o m i t a n t with the appearance in the reduced form (ferrocyt. b) of a high p K a group (m) and accompanied by its protonation (H +-ferrocyt. b ) - - a n d further oxidation at high potential ( e - ) - - c o n c o m i t a n t with the simultaneous disappearance of the high pKa group of the reduced form and the reappearance of a high pK* group (A) in the resulting energized oxidized form, which, in consequence, retains the previously fixed proton, although in a different position (ferricyt. b*-H+). (2) Direct protonation (H+ *) at low pK~ (zx). The five electrons of the ferric ion have been provisionally located in the three d orbitals of the lower energy-level, which is separated from the upper energy level of the other two d orbitals by ligand-field splitting energy.

222

is probably the real explanation of its uncoupling effect at the molecular level. Moreover, Fig. 23 summarizes schematically the above described reversible operation of chloroplast cytochrome b-559 at two alternate midpoint potentials and two alternate pK~'s, i.e., its simultaneous function as a transducing redox system and as a transducing acid-base system. Energy-coupling between both systems occurs unavoidably because both share a common intermediate which exists either in its electronically energized state (energized oxidized form = ferricyt, b*) or in its stabilized basal state (unenergized oxidized form = ferricyt, b). The proposed scheme also takes into consideration the existence of two energy levels, separated by the ligand-field splitting energy, for the d orbitals of the cytochrome iron. Localization in these orbitals of the five ferric ion electrons is not only provisional but also irrelevant in principle, since only the entrance (or removal) of the sixth electron in (or from) an upper or lower orbital has to do with the aspects being considered. Although the subject is still very controversial (cf. Refs. 5, 9 and 10), it may be accepted (see Fig. 14) that cytochrome b-559 is located between the reduced side of photosystem II (plastoquinone) and the oxidized side of photosystem I (cytochrome f ) , precisely one of the sites of the photosynthetic non-cyclic electron transport that is coupled to photophosphorylation. We believe, therefore, that it is reasonable to propose (Fig. 24) that the unenergized ferricytochrome b (ferricyt. b) accepts an energy-rich electron (e-*) at low potential (LUg) in an upper orbital, becoming concomitantly protonated at high pK, by an energy-poor proton (H +). The resulting protonated ferrocytochrome (H+-ferrocyt. b) is subsequently oxidized at high potential (HU~*), viz., a low-energy electron (e-) is removed from a low-lying orbital. Finally, the energized and protonated ferricytochrome (ferricyt. b*-H ÷) dissociates an energy-rich proton (H +*) at low pK a. In this way a proton is translocated and increases its concentration at the expense of an electron that decreases its pressure. It should be added finally that light-induced proton transloca-

LU'o

H+ f e r r o c y t , b

"=

U o = 9 5 mV

~/~.

-

ferricyt b

"

7.6

H +I~)

e- .=

H+-ferrocyt.b

/'J°~ = 3 4 0

rnV

ferricyt. L~~ - H +

e-,~H +

<

-.

H+ ~<-

H+

Fig. 24. Transduction of redox energy into electronic energy and subsequently into acid-base energy by the chloroplast cytochrome b-559 system. In a first stage, the oxidized basal form (ferricyt. b) can accept electrons ( e - * ) at a low potential (LUg, pH 7,6 = 95 mV), the resulting reduced form becoming simultaneously protonated (H +) at a high pK~ (H+-ferrocyt. b). In a second stage, the reduced and protonated cytochrome can donate electrons ( e - ) at a higher potential (HU~*), the resulting oxidized and electronically energized form retaining its translocated proton (ferricyt. b * - H + ) . In a final stage, the energized and protonated ferricytochrome can dissociate its proton (H +*) at aqower p K . , becoming simultaneously deenergized to its original basal form.

223

tion by chloroplast preparations has been found [38] to be directly correlated with the proportion of cytochrome b-559 in its high-potential form (Fig. 25). The peculiar redox and acid-base properties of cytochrome b-559 seem to be shared by another chloroplast cytochrome of the energized oxidized-form type, cytochrome b-563, or cytochrome bo, which likewise may be involved in photophosphorylation, but of the cyclic type [1,6,7]. Recently, Malkin [39] observed that the midpoint potential of the high-potential form of cytochrome b6 is about 0 mV and independent of pH in the pH range from 6.8 to 9.0, whereas previously Fan and Cramer [40] reported that the midpoint potential of the low-potential form of this cytochrome is about -180 mV and dependent on pH in the pH range from 6.0 to 8.0. Moreover, the low-potential form of yeast mitochondrial cytochrome b-564 is pH-dependent, whereas the high-potential form is pH-independent [29]. Although the cytochrome a 3 component of cytochrome c oxidase belongs to the energy-transducing redox systems of the energized reduced-form type, it transforms redox energy into acid-base energy with the same net result as the b-type transducing cytochromes. In both types of systems, the intermediate which couples redox energy with acid-base energy seems to be the cytochrome itself, in either its reduced form (cytochrome a3) or its oxidized form (cytochromes b). As shown schematically in Fig. 26, the corresponding intermediate participates cyclically in the energy-transducing process by alternating between its electronically energized state and its unenergized basal state. It was mentioned in the preceding section that flavins can not only transduce light energy into redox energy but also into acid-base energy, thus behaving

100

O~

75 e50

o 5

50 1100

=

C0rretation index = 0.983

25

0

0'.2

0'.4

o'.e

0'.8

Protons translocated (//mole H+/mg of chlorophyll)

Fig. 25. Light-induced proton translocation by spinach thylakoid preparations is directly correlated with the proportion of cytochrome b-559 in its high-potential form (HP). This proportion decreased gradually from a maximal value (expressed as 100 %) when the thylakoid suspensions were treated for 5 rain with increasing concentrations (expressed in p.p.m, along the line) of the detergent Triton X-100 (cf. Ref. 38).

224

+% oJ

bJ e_ .~=,/

~

ferricyt.b ~ - " "

~H

+

e- - I "

~

ferrocyt.a3 " j

~ H+

Fig. 26. Coupling between redox energy and acid-base energy through electronic energy by either a b-type cytochrome (energized oxidized-form type) or cytochrome a 3 (energized reduced-form type). In the first case (left), an energy-rich electron (e *) would reduce at a low potential the cytochrome b in its basal oxidized state (ferricyt. b) and would be removed at a higher potential (e-), leaving the cytochrome again oxidized and electronically energized (ferricyt. b*). During the redox process a proton (H ÷) would be fixed at a high pK* by the energized ferricytochrome b, which would afterwards dissociate it at a lower p K a (H + *), becoming simultaneously deenergized to its original oxidized basal form. In the second case (right), an energy-rich electron (e- *) would reduce at a low potential ferricytochrome a to its electronically energized reduced form (ferrocyt. a~'). During the redox process a proton (H +) would be fixed at a high pK,, by the energized ferrocytochrome a3, which would afterwards dissociate it at a lower pK* (H+*), becoming simultaneously deenergized to its basal form (ferrocyt. a3). Finally, the unenergized ferrocytochrome would be oxidized at high potential ( e - ) to its original oxidized form.

simultaneously as photosystems of the energized oxidized- and basic-form type. It is therefore very likely that flavins can also transduce redox energy into acid-base energy of vice versa [2-4,8]. The following reasons support our claim: (1) Flavins increase their midpoint redox potential not only upon excitation of their unenergized oxidized form by illumination, but also upon energization by protonation at low pH, as pointed out by the following equations: Low Ut;

FN+e-*

FN ~ ~

High U'~

FN ~ FNH"

~

FN* + e

High U'~' ~ FNH++e-

(2) Flavins increase their pK~ not only upon excitation of their unenergized basic form by illumination, but also upon reduction at low potential, as pointed out by the following equations: Low pK.

FNH +

~

FNH +

FN + H +*

High pK* ~ FN* + H +

High pK~

FNH"

~

FN ~ + H +

225

(3) Some flavoproteins, such as dihydrolipoyl dehydrogenase or ferredoxin reductase, exhibit midpoint redox potentials at the pyridine nucleotide level, whereas others, such as succinate dehydrogenase or fatty acyl-CoA dehydrogenases, exhibit midpoint redox potentials at the ubiquinone level. (4) NADH-dehydrogenase, the FMN-containing enzyme complex in energy-conserving site 1 of the respiratory chain, transfers electrons from the pyridine nucleotide level to the ubiquinone level, and is therefore, in our opinion, a prime candidate to behave, like cytochrome b-565 and cytochrome a 3, as a redox-linked vectorial proton pump by functioning at two alternate redox potentials and two alternate pKa's. As shown schematically in Fig. 27, unenergized oxidized flavins (FN) are compulsorily reduced at low potential (LUg) and become concomitantly protonated

R

R

I

_

I

0

~

ll

0

IL

+

o._ +

R I

OH

R IH

H I

H+

0

N÷ Fig. 27. Flavins are energy-transducing systems of both the energized oxidized- and basic-forrn type, which can operate at either two alternate midpoint redox potentials (LU~ and HU~*) or two p K , ' s (LpK,, and HpK*). Reduction ( e - *) at low potential (LUg) of the basal oxidized form (FN) implies protonation (H +) at pH's below the high pK~ of the group at position 4 (I), proper to the reduced form ( F N - ) . Oxidation ( e - ) at high potential (HU~*) of this protonated and reduced form ( H + - F N 7) implies proton translocation from the high p K a group of the reduced form to the high pK,, group at position 1 (v), proper to the energized oxidized form ( F N * - H + ) . On the other hand, direct protonation (H +*) of the basal oxidized form can only occur at p H ' s below its low pK,~ (v) and is concomitant with its electronic energization. A high-energy orbital and a low-energy orbital have been schematically represented below the corresponding structural formula to facilitate comprehension of the redox and acid-base reactions involved.

226

(H+-FN~), since reduction implies the appearance of a group of high pK, around 8 [41] at position 4. Oxidation at high potential (HU~*) of the reduced and protonated flavin implies the disappearance of the high pK, group at position 4 of the reduced form and the simultaneous reappearance of a high pK* group at position 1 of the resulting electronically energized form (FN*-H+). Oxidation-reduction at high potential of the protonated flavin thus involves proton translocation from position 1 to position 4 or vice versa. Proton dissociation of the energized oxidized flavin can occur at high pK* (not shown), or at low pK a, depending on whether the resulting oxidized and unprotonated form remains electronically energized or becomes stabilized into its original basal state. It should also be noted that protonation of the unenergized oxidized flavin, which is characterized by its low pK,, can only occur at low pH and brings about its electronic energization. We would, in conclusion, propose that NADH-dehydrogenase can operate as a redox-linked proton pump at energy-conserving site 1 of the respiratory chain, according to the scheme presented in Fig. 28. Unenergized oxidized flavin (FN) accepts an energy-rich electron (e-*) at a low potential (LUg) of about -230 mV at pH 7 [42] in an upper orbital and becomes concomitantly protonated at high pK, in position 4 by an energy-poor proton (H+). The resulting protonated semiquinone ( H + - F N -) is subsequently oxidized at high potential (HUd*) by removal of an energy-poor electron (e-) and becomes electronically energized and protonated in position 1. Finally, the energized, oxidized and protonated flavin (FN*-H +) can either dissociate at high pK* its proton (not shown), keeping itself energized (FN*), or, alternatively, as shown in the figure, dissociate at low pK, its proton (H +*) by stabilizing its electronic configuration and returning to its original basal form. As in the case of transducing cytochromes, a proton is translocated, increasing its concentration at the expense of an electron that decreases its pressure.

H+_FNE U ' o = - 2 9 0 mV p Ka

FN

~-

e-~H +

= 8

e_ .

H +*

e-

H+

Q-

HU~" !

e-~,l

H+FN"

Uo*= + 3 1 0 m V

FN

.

-H

+

Fig. 28. Transduction of redox energy into electronic energy and subsequently into acid-base energy by a flavin system. In a first stage, the oxidized basal form (FN) can accept electrons (e- *) at a low potential (LUg, pH 8, - 2 9 0 mV), the resulting reduced form becoming simultaneously protonated (H +) at a high pK,, ( H + - F N ~ ) . In a second stage, the reduced and protonated flavin can donate electrons ( e - ) at a higher potential (HU~*), the resulting oxidized and electronically energized form retaining its translocated proton ( F N * - H + ) . In a final stage, the oxidized and protonated flavin can dissociate its proton (H ÷ *) at a lower p K a, becoming simultaneously deenergized to its original basal form.

227 COUPLING BETWEENACID-BASE ENERGY AND PHOSPHATE-BONDENERGY As mentioned in the preceding section, it has as yet not been possible to solve the molecular mechanism of ATP synthesis by the respiratory or photosynthetic F0F1ATPase driven by the energized protons, generated during electron transport, when they flow back through that enzyme complex embedded in the mitochondrial or thylakoid membrane [26,43]. Proton translocation is, however, a fundamental property that FoF:ATPases of bacteria, mitochondria and chloroplasts share with another kind of ATPase, recently identified in the plasma membranes of fungi and higher plants [44]. This protontranslocating plasma membrane ATPase of plant cells shares, moreover, with several cation- and proton-translocating ATPases of animal cells the very significant property of forming a phosphorylated intermediate, namely, a mixed anhydride between phosphate and the/~-carboxyl group of an aspartate residue in the polypeptide chain of the enzyme [45-48]. When all these facts are brought together, they may have important implications for the mechanisms of respiratory and photosynthetic phosphorylation at the expense of a proton gradient, on one hand, and of ion translocation, in general, at the expense of ATP, on the other. If our reasoning could be extended to energy coupling between acid-base and phosphate-bond systems, the crucial requirement would be for a common intermediate able to occur both in an electronically energized state and in a stabilized basal state. As discussed before, a carboxylate anion would certainly be a prime candidate for this function, since it can exist either unenergized ( R - C O O - ) , or energized one ( R - C O O - ) * , like a carboxylate anion, or monomeric anhydride of a carboxylic acid, or energized twice as an acylium cation (R-C=O)**. With regard to phosphate, it would also fulfill the requirement, since it can exist either unenergized (Pi = HOPO~-), or energized one as a monomeric anhydride of phosphoric acid ( - O P O 2 - ) *, or energized twice as a metaphosphate monoanion or phosphonium group ( - P = P O f = + PO 2-)**. The acylium cation might be transitorily protected by a thiol group as a thioester. In this respect, the results of Griffiths et al. [49] suggesting that an unknown non-phosphorylated derivative of lipoic acid may participate in some way in the capture and transmission of energy deserve consideration. According to this interpretation, a carboxylate anion would be first electronically energized by the sequential action of two energy-rich protons (H + *) to an acylium cation. Whether the generation of this acylium cation would take place with the cooperation of two carboxylate groups and the transitional formation of an anhydride is, in principle, irrelevant. However, it deserves to be underlined, as mentioned above, that energization of the carboxylate anion should occur simultaneously with the removal from it, as water, of an oxide anion, as pointed out by the equation: R - C O O - + 2 H +* ~ ( R - C = O ) * * + H 2 0 The electronic energy stored in the acylium cation would then be transferred,

228

through the intermediate formation of an acyl-phosphate, to inorganic phosphate, which as a consequence of its energization would dissociate its otherwise very tightly bound proton: ( R - C = O ) * * + Pi ~ R - C O 0 - +

- P** + H +

In summary: 2 H +* + Pi ~ H 2 0 + -- P** + H+ It must be noted, however, that although the net result of the overall process is the removal by two energy-rich protons of an oxide anion from an inorganic phosphate molecule which dissociates an energy-poor proton, it is theoretically very unlikely that such a process may occur directly without the participation of an intermediate pair, similar or identical to the carboxylate anion/acylium cation just discussed. For one simple reason this statement seems to be valid: inorganic phosphate in its basal state exhibits two basic groups with pKa's of about 7 and 2 that would merely become protonated at low pH. Notwithstanding this, the fact remains that whatever the intermediate may be, it certainly is elusive and resists isolation. Once inorganic phosphate has become doubly energized it can transfer with itself its electronic energy to A D P for the synthesis of ATP: - P** + A D P ~ A D P * - O - P * The reverse overall process, namely, ATP-driven proton translocation catalyzed by ATPase, would eventually imply the dissociation of two energy-rich protons from water by a twice-energized phosphonium group from ATP. The resulting oxide anion as well as an energy-poor proton would eventually be fixed by phosphate in its unenergized form, according to the equation: - P**

+ H20 + H+~

Pi + 2 H + *

As stated above, it is most likely that the phosphonium group exerts its dissociating

2H

R-C:0] >

c t~d

H20 ~

xRTCO0--

~"

Pi

Fig. 29. Schematic representation of a possible coupling mechanism between acid-base energy and phosphate-bond energy through electronic energy. Two energized protons ( H - * ) would deenergize themselves by removing, as water, an oxide anion from a carboxylate anion ( R - C O O - ) , which would become concomitantly energized to an acylium cation (R-C=O)*. Subsequently, the acylium cation would transfer its electronic energy to inorganic phosphate (Pi~, which would become energized to a phosphonium group ( - P*) and dissociate simultaneously its very tightly bound proton. In this way, the acylium cation would deenergized itself to the original carboxylate anion in its basal state, closing the cycle.

229

action on water through either an intermediate carboxylate anion/acylium cation pair or a similar one. Figure 29 shows schematically how the carboxylate anion/acylium cation pair could participate cyclically in energy coupling between acid-base and phosphatebond systems. An oxide anion would be removed from the carboxylate anion by two energy-rich protons, with the concomitant formation of water and the energization of the carboxylate anion to an acylium cation. Subsequently, the acylium cation would transfer its electronic energy to inorganic phosphate, which would become energized to a phosphonium group and dissociate its proton as an energy-poor one. In this way, the acylium cation would de-energize itself to the original carboxylate anion in its basal state, closing the cycle. We would like to conclude this article with the following relevant consideration: What seems to be the essential feature of the phosphorylating mechanism--either at the substrate level (see p. 207) or the membrane level--is the conversion of the stable tetrahedral configuration of the ortophosphate anion into the unstable trigonal configuration of the metaphosphate anion through the more or less direct removal from Pi--either by two energized electrons or by two or three energized p r o t o n s - - o f an oxide anion and an energy-poor proton, i.e., an hydroxide anion. The oxide or hydroxide anion might be separated from the phosphorus atom either by direct pulling (e.g., with an acylium cation) or indirectly by pushing on the three other oxygens of the ortophosphate molecule (e.g., with three protonated carboxylate anions). ACKNOWLEDGEMENTS

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