Respiratory Electron Transfer Complexes
13. Roualt, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell (Cambridge, Mass.) 64, 881-883 14. Theil, E. C. (1990) J. Biol. Chem. 265,477 1-4774 15. Klausner, R. D. & Harford, J. B. (1989) Science 244, 357-3 59 16. Huynh, B. H., Moura, J. J. G., Moura, I., Kent. T. A., LeGall, J.? Xavier, A. V. & Munck, E. (1980) J. Riol. Chem. 255,3242-3244 17. Moura, I., Moura, J. J. G., Munck, E., Papaefthymiou, V. & LeGall, J. (1986) J. Am. Chem. SOC. 108, 349-351 18. Surerus, K. K., Munck, E., Moura, I., Moura, J. J. G. & LeGall, J. (1987)J. Am. Chem. SOC.109,3805-3807 19. Conover, R. C., Kowal, A. T., Fu, W., Park, J.-B., Aano, S., Adams, M. W. W. &Johnson, M. K. (1990) J. Biol. Chem. 265,8533 20. Butt, J. N.. Armstrong, F. A., Breton, J., George, S. J., Thomson, A. J. & Hatchikian, E. C. (1991) J. Am. Chem. SOC.in the press 21. Bovier-Lapierre, G., Bruschi, M., Bonicel, J. & Hatchikian, E. C. (1987) Biochim. Biophys. Acta 913,20-26 22. Yates, M. G. (1970) FEBS Lett. 8,281-285 23. Armstrong, F. A., George, S. J., Cammack, R., Hatchikian, E. C. & Thomson, A. J. (1989) Biochem. J. 264, 265-273
24. George, S. J., Richards, A. J. M., Thomson, A. J. & Yates, M. G. (1984) Biochem. J. 224,247-25 1 25. George, S. J. (1986) Ph.D. Thesis, University of East Anglia, Nonvich 26. Armstrong, F. A., George, S. J., Thomson, A. J. & Yates, M. G. (1988) FEHS Lett. 234, 107-1 10 27. Armstrong, F. A., Butt, J. N., George, S. J., Hatchikian, E. C. & Thomson, A. J. (1989) FEBS Lett. 259, 15- 18 28. Thomson, A. J., Robinson, A. E., Johnson, M. K., Moura, J. J. G., Moura, I., Xavier, A. V. & LeGall, J. (1981) Biochim. Biophys. Acta 670,93-100 29. Hatchikian, E. C., Cammack, R., Patel, D. S., Robinson, A. E., Richards, A. J. M., George, S. J. & Thomson, A. J. (1984) Biochim. Biophys. Acta 784, 40-47 30. Johnson, M. K., Robinson, A. E. & Thomson, A. J. (1982) in Iron-Sulfur Proteins (Spiro, T. G., ed.), vol. 4, chapter 10, Wiley, New York 31. Armstrong, F. A,, Cox, P. A,, Hill, H. A. O., Lowe, V. J. & Oliver, B. N. (1987) J. Electroanal. Interfacial Electrochem. 217,331-366
Received 18 April 1991
Electron transfer in succinate :ubiquinone reductase and quinol :fumarate reductase J. C. Salerno Biology Department, Rensselaer Polytechnic Institute, Troy, NY I 2 180, U.S.A.
Introduction Succinate :ubiquinone reductase (SQR) catalyses the oxidation of succinate to fumarate, resulting in the reduction of ubiquinone to ubiquinol. In mitochondria and aerobic bacteria, ubiquinol donates electrons through an electron transfer system to molecular oxygen, producing water, and driving the synthesis of A T P through the creation of a proton electrochemical gradient. In some bacteria growing with fumarate as the terminal oxidant, menaquinol produced via the oxidation of low-potential substrates in turn reduces fumarate to succinate to a reaction catalysed by menaquinol :fumarate reductase (QFR). This also results in the generation of a proton electrochemical gradient of the same sign as that produced by succinate oxidation. Considering the vectorial nature of energy conservation, it is Abbreviations used: SQR, succinate:ubiquinone reductase; QFR, menaquino1:fumarate reductase; FP, a 70 kDa flavoprotein; IP, 30 kDa catalytic subunit; Qs, bound ubisemiquinone.
clear that the QFR system is not merely a version of the SQR system kinetically optimized to run in the reverse direction. SQR and QFR have been the subject of a number of recent reviews [ 1-31. Both consist of two large, relatively soluble subunits, often termed the catalytic subunits, and one or two smaller hydrophobic subunits. T h e catalytic subunits consist of FP, which is a flavoprotein of molecular mass of about 70 kDa and contains the succinate/fumarate catalytic site, and IP, which contains three ironsulphur clusters and has a molecular mass of about 30 kDa. These subunits are highly homologous in SQR and QFR enzymes. T h e hydrophobic anchor polypeptide components are not conserved between systems. In Escherichzh coli both SQR and FQR enzymes can be synthesized; both have two anchor polypeptides of similar size (about 14 m a ) , but n o significant sequence identity exists. In E. coli, the hydrophobic peptides of SQR carry a low-potential b-type cytochrome. No such cytochrome is associated with the QFR enzyme in
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E. coli, but in other fumarate reductases a b cyto-
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chrome does exist. In other organisms, SQR may have a single large anchor polypeptide and may have two associated b haems. In both SQR and QFR the anchor polypeptides also appear to contain the sites of quinol/quinone binding. The iron-sulphur centres of both SQR and QFR were discovered initially by low temperature e.p.r. In SQR, centres S-1 and S-3 are reduced by succinate in equilibrium experiments, while centre S-2 has a low apparent mid-point potential. The situation is similar in QFR, except that FR-3 has a slightly lower potential than FR-1. Centre S-1, and the corresponding centre FR-1 in the fumarate reductase system, is a 2Fe-2S* cluster. The discovery of 3Fe-4S* clusters in bacterial ferredoxins opened the door to work which showed that centre S-3 (and centre FR-3) is a three-iron cluster and that the two low-potential clusters are four-iron clusters [4]. While the role of the FAD cofactor and the two succinate reducible iron-sulphur clusters is widely accepted, the role of the low-potential prosthetic groups has been controversial. In SQR, the stabilized semiquinone states of the FAD and associated ubiquinones provide ideal n = 1/ n = 2 converters; this suggested a model in which electron transfer through SQR was basically sequential, in the order succinate, flavin, S-1, S-2, S-3, UQ [5]. Magnetic interactions suggesting proximity between flavin and S-1, between S-1 and S-2 and between S-3 and bound ubisemiquinone were known, and other evidence suggested interactions between S-2 and S-3. In the QFR system, weak interactions between FR- 1 and FR-3 were discovered; features of the e.p.r. spectra of both SQR and QFR were later proposed to indicate strong interactions between S-2 and S-3 [6-91. The low apparent potential of S-2 was initially proposed to result from interactions, possibly of an electrostatic nature, between S-1 and S-2 [9a]. Cammack and co-workers have proposed several modifications of this picture of QFR and SQR [6, lo]. In place of a sequential model, they proposed a dual pathway model in which electron pairs from FADH, would be split. The first electron, with a higher potential, would reduce S-1, while the second, lower potential electron, would reduce S-2. S-1 would reduce S-3 while S-2 reduced the low potential b cytochrome; S-3 and the b cytochrome would in turn reduce ubiquinone. In order to prevent electron transfer between the high- m d low-potential pathways, the catalytic cycle would always have to begin in an odd electron state,
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which in their proposal could always be regained by the release of a semiquinone. A related proposal by the same workers attributes the four line e.p.r. spectrum of the bound ubisemiquinones (Qs) associated with SQR to an interaction between a single bound semiquinone and low potential cytochrome b. Ruzicka et al [ 1I] had attributed the four line spectrum to dipolar coupling between two nearly isotropic species. Computer simulations of their experimental spectrum gave a reasonable fit to the data from the assumption that the interacting species were a semiquinone radical and centre s-3; an even better fit was obtained for interactions between two radicals. Later, redox titrations of the signals eliminated FAD radicals as a potential partner in the interaction [ 121. T o fit the unusually sharp titration of the semiquinone signal, it was necessary to assume that both species had similar bell-shaped titration profiles; in this case, the titration of the signal owing to the interaction of the two semiquinones was the square of the titration of either alone [13]. The rapid relaxation of the Qs split signal and the unsplit free radical also observed was attributed to interactions with nearby transition metals, with centre S-3 and cytochrome bS6"the most likely candidates.
Simulation of magnetic resonance spectra The identification of Qs as an ubiquinone pair thus rests on simulation of both magnetic resonance data and simulation of redox titration data, as well as on quinone extraction and replenishment experiments which prove that at least one of the partners must be ubisemiquinone. Turning to the possibility that the other partner could be a very anisotropic species such as a low-spin ferrihaem, we first examine the origin of the four lines in the spectrum simulated by Ruzicka et al. [ 111. Dipolar coupling has a characteristic 1-3cos2# angular dependence, where # is the angle between the magnetic field and the vector connecting the two spins. In a frozen solution, all angles are present and the spectrum is the sum of contributions from all orientations. The angular term has its greatest absolute value with # = 0; the angular term is then equal to - 2. This gives rise to features spaced 2 0 from the centre of the four line pattern, which is the position of the resonance in the absence of coupling. D is proportional to the product of the magnetic moments and inversely proportional to the cube of distance; for two nearly isotropic species, such as quinone radicals, D itself has no significant angular dependence. The most probable absolute value of the angular
Respiratory Electron Transfer Complexes
term is one; orientations for which this is true give rise to the strong pair of inner lines in the spectrum of two dipolar-coupled radicals. As shown by previous workers, the fit of the experimental spectrum to the four lined simulation is impressive. In Fig. 1, the top trace is a simulation of such a four line spectrum caused by the interaction of two isotropic radicals with g= 2.00 and linewidth of 1.2 mT. The value of D was taken as 3.0 mT, which corresponds to a distance slightly less than 0.8 nm. In the following three traces, the simulations depict spectra resulting from the splitting of a radical by a low-spin ferrihaem with g values of 3.4, 1.7 and 0.9, which should be a reasonable approximation of the g-tensor of cytochrome bshOThe traces correspond to orientation of the interspin vector along the maximum g value of the haem, along the minimum g value of the haem, and along the direction equidistant from the principal axes of the g-tensor coordinate system. Owing to the well known ‘3/2’ effect which reduces the effect of dipolar coupling between unlike spins by rendering the off diagonal matrix elements of the dipolar Hamiltonian ineffective, reproducing the splittings approximately would necessitate reducing the spin-spin distance from about 0.77 to about 0.68 nm. The details of the spectrum are not well reproduced for any choice of parameters because the angular dependence of the ferrihaem g-tensor, applied through D,is overlayed on the angular dependence of the dipolar coupling. In Fig. 2, the top trace is a simulation of dipolar coupling between the same ferrihaem and radical with the interspin vector along the direction of the g-tensor co-ordinate system corresponding to the intermediate g value. In this case the outer lines do not correspond to a unique direction, but result from orientations in which either orientation corresponding to the largest or to the intermediate haem g value is along the applied field direction. Spectra of the split signal in oriented multilayers are consistent with dipolar coupling between radicals in which the interspin vector is along the normal to the membrane plane, but would not be consistent with the angular dependence of spectra such as this. The lower trace of Fig. 2 represents the use of a simulation with both isotropic exchange and dipolar coupling between a haem and a radical to obtain a four line pattern with the same spacing as the initial spectrum, which resulted from dipolar coupling alone between two radicals. Exchange interactions could not significantly contribute to the splitting in the case of the two coupled radicals, because they are nearly isotropic and hence can differ in resonant field bv no more than a few tenths __ . I
Fig. I
Computer simulations of e.p.r. spectra of spincoupled pairs Field range is 0.31 t o 0.33 mT and resonant frequency is 9.1 GHz. Parameters for spectra are, in order from top. ( a ) Dipolar-coupled radical spectrum, g values isotropic at 2.0, linewidths I .2 mT, D = 3 mT. ( b ) Dipolar-coupled radical and slowspin ferrihaem, D = 4.5 mT, haem g, = 3.4, g, = I .7, gy= 0.9 with interspin vector along z axis. (c) As above, with interspin vector axis along x axis. ( d ) As above, with interspin vector equidistant from x , y and z axes.
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Magnetic field
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Fig. 2
Computer simulations of haem-radical-coupled pair as in Fig. I.
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Top: interspin vector along y axis. Bottom: with dipolar and exchange terms to produce four line spectrum; interspin vector along z axis, D = 6.6 mT, J = 2.4 mT.
0.01 T
V Magnetic field
of a mT. Note that the outer lines of the upper trace of Fig. 1 are about twice as intense relative to the inner lines as is the case in the lower trace of Fig. 2. The reason for the low intensity of the outer lines is that the additional orientation dependence introduced by the g-tensor anisotropy of the haem is still in effect; the splitting in this case therefore varies much faster with angle than in the dipolar-coupled biradical, and many fewer spins lie near the extrema. It appears to be impossible to simulate the e.p.r. spectra assuming coupling between the haem and a radical. Since both the orientation dependence of the split signal and the redox titration data argue for a ubisemiquinone pair, which accounts well for the observed spectra, it does not appear feasible to substitute a haem for one of the radicals. On the other hand, it is clear that the Qs site is intimately associated with cytochrome bS6@Only SQR or succinate :cytochrome c reductase (SCR) preparations which contain high levels of the
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cytochrome exhibit the split signal. SQR preparations isolated or reconstituted under conditions in which cytochrome bshOis low exhibit a semiquinone radical which relaxes faster than the magnetically isolated Qi radical of the quino1:cytochrome c reductase complex, but is much slower relaxing than the Qs radical in more intact systems [14]. This suggests that the haem is indeed providing part of the relaxation pathway for the radical; the additional rapid relaxation presumably arises from centre S-3. It also appears that the integrity of the Qs site depends on the presence of the haem, which is believed to cross-link the two anchor polypeptides. Yu etal [ 151 showed that the e.p.r. spectrum of the haem reversibly and dramatically shifted upon dissociation of the two soluble subunits. The e.p.r. and optical spectra of the haem are sensitive to the binding of inhibitors which block electron transfer from centre S-3 to quinone; Kd values determined for such inhibitors using the red shift of the optical spectra of cytochrome bshOwhich they produce are comparable with K, values for inhibition of electron transfer [ 141. It is remarkable that the SQR system, which should not, from a chemiosmotic standpoint, require a membrane-inserted electron-carrying arm, has a cytochrome component, while fumarate reductase, which should require transmembrane electron transfer for chemiosmotic function, is devoid of such a component in the E. coli system. In fumarate reductases which have associated cytochromes, Garlands suggestion that they represent a transmembrane electron carrying arm remains attractive. The b haem in the E. coli SQR may have a vestigal structural role; the Qs pair may function to move reducing equivalents and protons into the bilayer. It is possible that the menaquinone binding site in the E. coli QFR system is connected to the surface via a channel, but it is also possible that two or more Q binding sites are present. If this is the case, arrangement of the Q sites to span the membrane, or at least enough of it to bridge the barrier to protonic movement, would increase the Q-Q distance to the point where dipolar coupling would be too weak to produce splittings. This would also render direct proton transfer between quinones unlikely, while electron transfer, which can proceed over large distances, might be catalytically competent. The low potential of centre S-2 has long been a point of contention between workers in the field; for years many laboratories found it difficult to accept the existence of such a low-potential com-
Respiratory Electron Transfer Complexes
ponent in a system reduced by a 0 mV substrate couple. Centre S-2 is not well isolated from other components, however. Interactions between centres S-1 and S-2 have been established for years. Previously, simulations of the splitting of the e.p.r. spectrum of centre s-1 by centre s-2 were performed assuming identical g-tensors but different orientations and suggested that the distance between them was 1-1.2 nm [9a]. Fig. 3 shows similar simulations of the splitting of the central resonance of centre S-2 as in the previously published spectra of reconstitutively inactive preparations of succinate dehydrogenase. Such preparations lack centre S-3, which also interacts with centre S-2 and modulates the S-1/S-2 interaction; they are therefore more easily approached. The top trace shows a simulation in which dipolar coupling consistent with 1 nm separation and 0.2 m T of exchange coupling were Fig. 3 Simulation of splitting of the e.p.r. spectrum of S- I by centre S-2 Field range is 0.30 t o 0.36 mT. Top trace shows dipolar- and exchange coupling of I and 0.2 mT respectively. g values (S-I) 2.03, 1.93, 1.905 (S-2) 2.06, 1.92, 1.85. lnterspin vector oriented 62” (e), 85” Lower trace shows spectral simulation without interaction terms.
(I).
Magnetic field
used; the lower trace shows the simulated spectra of S-1 and S-2 in the absence of coupling. More detailed simulations may bring out additional details, but clearly the distance and orientation with respect to the S-1 cluster g-tensor co-ordinate system deduced from the previous simulations are approximately correct. More recently, it has been proposed that interactions between S-2 and S-3 account for the wings of the e.p.r. spectra in succinate dehydrogenases and fumarate reductases in the fully reduced state. These wings are indicative of much closer coupling between S-2 and S-3 than between S-1 and S-2. Because of the proximity of these centres, it is of dubious validity to treat them as thermodynamically independent entities in analysing the thermodynamics of electron transfer.
Thermodynamics and mechanism The flavin semiquinone in the SQR system is only slightly unstable at pH 7 . Furthermore, both the FADHJFADH and FADH/FAD couples (E”’= - 30 and - 130 mV, respectively) are capable of reducing centre S- 1 without difficulty; the two electron reduction of flavin by succinate is slightly endergonic but can be readily pulled by electron transfer to the higher potential iron-sulphur centres. The uphill two electron step is not helped by division of the following pathway into high- and low-potential arms. The bound semiquinones at the Qs sites associated with SQR are even more stabilized, having at physiological pH stability constants of order unity. These bound quinones are nearly isopotential with centre S-3. The Qs site is thus a highly effective n = l / n = 2 converter well suited to facilitating reduction of ubiquinone to ubiquinol by centre S-3 in two sequential one electron steps. In the QFR system in E. coli, the stability of both the flavosemiquinone radical and a menasemiquinone radical are comparable with the stability of flavosemiquinone in SQR, the two electron potentials for these species are about - 12 mV and - 80 mV respectively (D. Simpkin & W. J. Ingledew, unpublished work). The fumarate reductase system thus is equipped with two potential n = l / n = 2 converters in much the same way as in SQR. As in the SQR system, the low-potential one-electron couples for both flavin and quinone are only 40-50 mV lower in potential than the two-electron couples for each; they are thus more nearly isopotential with the succinate reducible clusters than with the lowpotential centres s-2 or FR-2. If the potentials and stabilities of the radical species associated with QFR and SQR are not well
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suited to a dual pathway scheme for either enzyme, the question of the low potentials of centre S-2 and centre FR-2 remains. Earlier, my co-workers and I pointed out the possibility that interactions between S-1 and S-2 contributed to the low apparent midpoint potential of the latter centre, which might participate in electron transfer by being transiently reduced with an effective potential only 60-100 mV more negative than that of centre S-1 [9a]. Since then it has become clear that S-2 and S-3 are even more closely associated than S-1 and S-2. If we consider the possibility that s-1, s-2 and s-3 are arranged in that order spatially, which is consistent with the strengths of the magnetic interaction between them, we can model the titrations of the three iron-sulphur clusters by assigning interactions between S-1 and S-2 and between S-2 and S-3, and assuming that S-1 and S-3 do not significantly interact directly. We assume that from the oxidized enzyme one-electron reduction of S-3 has a midpoint potential of 60 mV; one-electron reduction of either S-1 or S-2 would have a potential of 0 mV. Interactions between S-2 and S-3 cause the midpoint of either to be lowered by 180 mV upon reduction of the other; an interaction between S-1 and S-2 causes a similar lowering of the mid-point of either by 60 mV upon reduction of the other. In an equilibrium titration, the dominant one-electron form of the enzyme would have centre S-3 reduced; only about 5% of the one-electron reduced enzymes would have centre S-2 reduced; since in these enzymes S-3 would be oxidized and hence odd electron, S-2 would be unlikely to be directly detectable. The dominant two-electron reduced state would have S-1 and S-3 reduced; only 1% of the two-electron reduced enzymes would have S-2 reduced, and in most of these S-3 would also be odd electron. As a result, the titration of S-2 would be observed as an almost ideal n = 1 species at - 240 mV; S-1 and S-3 would titrate as almost ideal n = 1 species at 0 and 60 mV respectively. The behaviour of the iron-sulphur centres in QFR can be as successfully simulated by assigning FR-2 and FR-3 potentials of - 60 mV and FR-1 a potential of 0 mV when all the other centres are oxidized; the interaction terms would be the same as in the SQR case. The result would be nearly ideal n = 1 titrations for FR-1, FR-2 and FR-3 at 0 mV, - 300 mV, and - 60 mV respectively. The potentials which would determine the function of the low-poteniial centres in electron transfer would be mechanism-dependent. Starting from a reduced resting state in which S-1 and S-3 were reduced and S-2 oxidized, reduction of centre
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S-2 by either centre would be unfavourable until S-3 was oxidized by quinol. At that point, the equilibrium constant for electron transfer between S-1 and S-2 would be unity. Once S-2 was reduced, reduction of S-3 would be favoured by a factor of ten. As soon as S-2 was oxidized by S-3, reduction of S- 1 could readily proceed. Starting from the oxidized state, electron transfer to S-1 from flavin would be followed by electron transfer from S-1 to S-2 with an equilibrium constant of unity, after which reduction of S-3 and reduction of S-1 by flavin could proceed. None of these steps need be significantly endergonic. In the same way, reduction of FR-2 by FR-3 in QFR could proceed with a microscopic equilibrium constant of unity even though in equilibrium titration experiments no significant reduction of FR-2 is observed with menaquinol or succinate as reductants. After the oxidation of FR-1 by flavin, FR-2 and FR-3 are essentially isopotential for the exchange of electrons. What are the sources of interactions between electron carriers which contribute to anti-co-operative behaviour? While conformationally-mediated interactions may contribute, electrostatic interactions are strong enough to account for terms of the magnitude needed to account for the observed behaviour. In free space, interactions between single electrons separated by 1 nm give an electrostatic potential energy term of 1.44 eV. Since the S-1 to S-2 distance is approximately 1 nm, an electrostatic term of 60 mV could be produced by a dielectric constant of 24. This is a very high dielectric constant compared with those usually used to represent the interior of proteins; water, with a dielectric constant of 80, is a much more effective quencher of electrostatic interactions. It is likely that the S-2 to S-3 distance is somewhat shorter than 1 nm; the potential due to a point charge is proportional to l/r, so the dielectric constant needed to produce 180 mV of interaction would probably be about 10. Since the two interaction terms could vary considerably (as long as their sum remained constant) and still account for the titration data, a uniform dielectric constant of 12-15 could provide the interactions needed. Given the proximity of the clusters, their location in a protein of substantial size, and the strength of electrostatic interactions over molecular dimensions, the real question is why larger interactions are not observed in proteins with multiple electron carriers. Bulk dielectric constants are not a replacement for the detailed calculations of electrostatic
Respiratory Electron Transfer Complexes
interactions which can be done when more structural information is available, but they do provide order of magnitude estimates of interaction strengths. Evidently, considerable rearrangement of charge driven by reduction significantly raises the effective dielectric. It seems likely that centres S-2 and FR-2 are apparently low-potential clusters merely because they are the middle clusters in the sequence of electron transfer. Interactions in the equilibrium state favour the separation of electrons; at lower levels of reduction, transient electron transfer through the intermediate cluster could be feasible without energetic cost. 1. Ohnishi, T. (1987) Curr. Top. Bioenerg. 15, 37-65 2. Cole, S. T., Condon, C., Lemire, B. D. & Weiner, J. H. (1986) Biochim. Biophys. Acta 135-381 3. Hedersted, I,. & Rutberg, I,. (1981) Microbiol. Kev. 45,542-555 4. Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, €3. A. C. & Kearney, E. B. (1985) J. Biol. Chem. 260,7368-7378 5. Ohnishi, T., King, T. E., Blum, H., Bowyer, J. R. & Maida, T. (1981)J. Biol. Chem. 256,5577-5582 6. Cammack, R., Crowe, B. A. & Cook, N. D. (1986) Biochem. Soc. Trans. 14, 1207-1208 7. Simpkin, D. & Ingledew, W. J. (1985) Biochem. SOC. Trans. 13,603-607 8. Johnson, M. K., Kowal, A. T., Morningstar, J. E.,
Oliver, M. E., Whittaker, K., Gunsalus, R. P., Ackrell, B. A. C. & Cecchini, G. (1988) J. Biol. Chem. 263, 14732-14738 8a. Johnson, M. K., Morningstar, J. E., Kearney, E. H. & Ackrell, B. A. C. (1988) in Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, B. & Ernster, I,., eds.), pp. 473-483, Plenum Press, N.Y. 9. Salerno, J. C. & Xu, Y. (1988) in Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, €3. & Ernster, I,., eds.), pp. 467-472, Plenum Press, N.Y. 9a. Salerno,J. C., Lim, J., King, T. E., Blum, J. & Ohnishi, T. (1979)J. Biol. Chem. 254,4828-4835 10. Cammack, R., Maguire, J. I. & Ackrell, B. A. C. (1988) in Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, B. & Ernster, I,., eds.), pp. 485-491, Plenum Press, N.Y. 11. Ruzicka, F., Beinert, H., Schepler, K. L., Dunham, W. K. & Sands, K. H. (1975) Proc. Natl. Acad. Sci. U S A . 72,2886 12. Ingledew, W. J., Salerno, J. C. & Ohnishi, T. (1976) Arch. Biochem. Biophys. 177, 176-184 13. Salerno, J. C. & Ohnishi, T. (1980) Biochem. J. 192, 769-78 1 14. Xu, Y., Salerno,J. C.. Wei, Y. H. & King, T. E. (1987) Biochem. Biophys. Res. Commun. 144, 123-128 15. Yu, I,., Xu, J. X., Haley, P. E. & Yu, C. A. (1987) J. Biol. Chem. 262,1137-1 143 Received 18 April 1991
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Bacterial dimethyl sulphoxide reductases and nitrate reductases
Alastair G. McEwan,* Neil Benson,* Tracey C. Bennett,* Steven P. Hanlon,* Stuart J. Fergus0n.t David 1. Richardson,t and J. Baz Jackson$ *School of Biological Sciences, University of East Anglia, Norwich, U.K.,+Department of Biochemistry, University of Oxford, Oxford, U.K. and $School of Biochemistry, University of Birmingham, Birmingham, U.K.
Introduction A wide variety of bacteria can respire using nitrate as an electron acceptor. Nitrate reductase catalyses the two electron reduction of nitrate to nitrite, and this reaction is the first step in denitrification. Nitrate reductases are, however, not restricted to denitrifying bacteria, and many enteric bacteria, for example, Escherichia coli, are capable of nitrate respiration [l]. In addition to their importance in the Nitrogen Cycle, bacteria also have a major role in the cycling of sulphur through their use of oxidized Abbreviations used: DMSO, dimethyl sulphoxide; DMS, dimethyl sulphide; nay, nitrate reductase gene; dms, DMSO reductase gene.
sulphur compounds as electron acceptors. Although the reduction of inorganic sulphur compounds such as sulphate is well established it is now also known that about 50% of the global flux of sulphur is in the form of organic sulphur, especially dimethyl sulphoxide (DMSO) and dimethyl sulphide (DMS) [Z]. DMSO reductase catalyses the two electron reduction of DMSO generating DMS, and over the last decade many bacteria which can respire using DMSO as an electron acceptor have been identified. These include photosynthetic bacteria of the genus Rhodobacter and enteric bacteria exemplified by E. coli A unifying property of nitrate reductases and DMSO reductases is that they are molybdenumcontaining enzymes [ 3 ] .However, there are signifi-
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13. Roualt, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell (Cambridge, Mass.) 64, 881-883 14. Theil, E. C. (1990) J. Biol. Chem. 265,477 1-4774 15. Klausner, R. D. & Harford, J. B. (1989) Science 244, 357-3 59 16. Huynh, B. H., Moura, J. J. G., Moura, I., Kent. T. A., LeGall, J.? Xavier, A. V. & Munck, E. (1980) J. Riol. Chem. 255,3242-3244 17. Moura, I., Moura, J. J. G., Munck, E., Papaefthymiou, V. & LeGall, J. (1986) J. Am. Chem. SOC. 108, 349-351 18. Surerus, K. K., Munck, E., Moura, I., Moura, J. J. G. & LeGall, J. (1987)J. Am. Chem. SOC.109,3805-3807 19. Conover, R. C., Kowal, A. T., Fu, W., Park, J.-B., Aano, S., Adams, M. W. W. &Johnson, M. K. (1990) J. Biol. Chem. 265,8533 20. Butt, J. N.. Armstrong, F. A., Breton, J., George, S. J., Thomson, A. J. & Hatchikian, E. C. (1991) J. Am. Chem. SOC.in the press 21. Bovier-Lapierre, G., Bruschi, M., Bonicel, J. & Hatchikian, E. C. (1987) Biochim. Biophys. Acta 913,20-26 22. Yates, M. G. (1970) FEBS Lett. 8,281-285 23. Armstrong, F. A., George, S. J., Cammack, R., Hatchikian, E. C. & Thomson, A. J. (1989) Biochem. J. 264, 265-273
24. George, S. J., Richards, A. J. M., Thomson, A. J. & Yates, M. G. (1984) Biochem. J. 224,247-25 1 25. George, S. J. (1986) Ph.D. Thesis, University of East Anglia, Nonvich 26. Armstrong, F. A., George, S. J., Thomson, A. J. & Yates, M. G. (1988) FEHS Lett. 234, 107-1 10 27. Armstrong, F. A., Butt, J. N., George, S. J., Hatchikian, E. C. & Thomson, A. J. (1989) FEBS Lett. 259, 15- 18 28. Thomson, A. J., Robinson, A. E., Johnson, M. K., Moura, J. J. G., Moura, I., Xavier, A. V. & LeGall, J. (1981) Biochim. Biophys. Acta 670,93-100 29. Hatchikian, E. C., Cammack, R., Patel, D. S., Robinson, A. E., Richards, A. J. M., George, S. J. & Thomson, A. J. (1984) Biochim. Biophys. Acta 784, 40-47 30. Johnson, M. K., Robinson, A. E. & Thomson, A. J. (1982) in Iron-Sulfur Proteins (Spiro, T. G., ed.), vol. 4, chapter 10, Wiley, New York 31. Armstrong, F. A,, Cox, P. A,, Hill, H. A. O., Lowe, V. J. & Oliver, B. N. (1987) J. Electroanal. Interfacial Electrochem. 217,331-366
Received 18 April 1991
Electron transfer in succinate :ubiquinone reductase and quinol :fumarate reductase J. C. Salerno Biology Department, Rensselaer Polytechnic Institute, Troy, NY I 2 180, U.S.A.
Introduction Succinate :ubiquinone reductase (SQR) catalyses the oxidation of succinate to fumarate, resulting in the reduction of ubiquinone to ubiquinol. In mitochondria and aerobic bacteria, ubiquinol donates electrons through an electron transfer system to molecular oxygen, producing water, and driving the synthesis of A T P through the creation of a proton electrochemical gradient. In some bacteria growing with fumarate as the terminal oxidant, menaquinol produced via the oxidation of low-potential substrates in turn reduces fumarate to succinate to a reaction catalysed by menaquinol :fumarate reductase (QFR). This also results in the generation of a proton electrochemical gradient of the same sign as that produced by succinate oxidation. Considering the vectorial nature of energy conservation, it is Abbreviations used: SQR, succinate:ubiquinone reductase; QFR, menaquino1:fumarate reductase; FP, a 70 kDa flavoprotein; IP, 30 kDa catalytic subunit; Qs, bound ubisemiquinone.
clear that the QFR system is not merely a version of the SQR system kinetically optimized to run in the reverse direction. SQR and QFR have been the subject of a number of recent reviews [ 1-31. Both consist of two large, relatively soluble subunits, often termed the catalytic subunits, and one or two smaller hydrophobic subunits. T h e catalytic subunits consist of FP, which is a flavoprotein of molecular mass of about 70 kDa and contains the succinate/fumarate catalytic site, and IP, which contains three ironsulphur clusters and has a molecular mass of about 30 kDa. These subunits are highly homologous in SQR and QFR enzymes. T h e hydrophobic anchor polypeptide components are not conserved between systems. In Escherichzh coli both SQR and FQR enzymes can be synthesized; both have two anchor polypeptides of similar size (about 14 m a ) , but n o significant sequence identity exists. In E. coli, the hydrophobic peptides of SQR carry a low-potential b-type cytochrome. No such cytochrome is associated with the QFR enzyme in
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E. coli, but in other fumarate reductases a b cyto-
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chrome does exist. In other organisms, SQR may have a single large anchor polypeptide and may have two associated b haems. In both SQR and QFR the anchor polypeptides also appear to contain the sites of quinol/quinone binding. The iron-sulphur centres of both SQR and QFR were discovered initially by low temperature e.p.r. In SQR, centres S-1 and S-3 are reduced by succinate in equilibrium experiments, while centre S-2 has a low apparent mid-point potential. The situation is similar in QFR, except that FR-3 has a slightly lower potential than FR-1. Centre S-1, and the corresponding centre FR-1 in the fumarate reductase system, is a 2Fe-2S* cluster. The discovery of 3Fe-4S* clusters in bacterial ferredoxins opened the door to work which showed that centre S-3 (and centre FR-3) is a three-iron cluster and that the two low-potential clusters are four-iron clusters [4]. While the role of the FAD cofactor and the two succinate reducible iron-sulphur clusters is widely accepted, the role of the low-potential prosthetic groups has been controversial. In SQR, the stabilized semiquinone states of the FAD and associated ubiquinones provide ideal n = 1/ n = 2 converters; this suggested a model in which electron transfer through SQR was basically sequential, in the order succinate, flavin, S-1, S-2, S-3, UQ [5]. Magnetic interactions suggesting proximity between flavin and S-1, between S-1 and S-2 and between S-3 and bound ubisemiquinone were known, and other evidence suggested interactions between S-2 and S-3. In the QFR system, weak interactions between FR- 1 and FR-3 were discovered; features of the e.p.r. spectra of both SQR and QFR were later proposed to indicate strong interactions between S-2 and S-3 [6-91. The low apparent potential of S-2 was initially proposed to result from interactions, possibly of an electrostatic nature, between S-1 and S-2 [9a]. Cammack and co-workers have proposed several modifications of this picture of QFR and SQR [6, lo]. In place of a sequential model, they proposed a dual pathway model in which electron pairs from FADH, would be split. The first electron, with a higher potential, would reduce S-1, while the second, lower potential electron, would reduce S-2. S-1 would reduce S-3 while S-2 reduced the low potential b cytochrome; S-3 and the b cytochrome would in turn reduce ubiquinone. In order to prevent electron transfer between the high- m d low-potential pathways, the catalytic cycle would always have to begin in an odd electron state,
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which in their proposal could always be regained by the release of a semiquinone. A related proposal by the same workers attributes the four line e.p.r. spectrum of the bound ubisemiquinones (Qs) associated with SQR to an interaction between a single bound semiquinone and low potential cytochrome b. Ruzicka et al [ 1I] had attributed the four line spectrum to dipolar coupling between two nearly isotropic species. Computer simulations of their experimental spectrum gave a reasonable fit to the data from the assumption that the interacting species were a semiquinone radical and centre s-3; an even better fit was obtained for interactions between two radicals. Later, redox titrations of the signals eliminated FAD radicals as a potential partner in the interaction [ 121. T o fit the unusually sharp titration of the semiquinone signal, it was necessary to assume that both species had similar bell-shaped titration profiles; in this case, the titration of the signal owing to the interaction of the two semiquinones was the square of the titration of either alone [13]. The rapid relaxation of the Qs split signal and the unsplit free radical also observed was attributed to interactions with nearby transition metals, with centre S-3 and cytochrome bS6"the most likely candidates.
Simulation of magnetic resonance spectra The identification of Qs as an ubiquinone pair thus rests on simulation of both magnetic resonance data and simulation of redox titration data, as well as on quinone extraction and replenishment experiments which prove that at least one of the partners must be ubisemiquinone. Turning to the possibility that the other partner could be a very anisotropic species such as a low-spin ferrihaem, we first examine the origin of the four lines in the spectrum simulated by Ruzicka et al. [ 111. Dipolar coupling has a characteristic 1-3cos2# angular dependence, where # is the angle between the magnetic field and the vector connecting the two spins. In a frozen solution, all angles are present and the spectrum is the sum of contributions from all orientations. The angular term has its greatest absolute value with # = 0; the angular term is then equal to - 2. This gives rise to features spaced 2 0 from the centre of the four line pattern, which is the position of the resonance in the absence of coupling. D is proportional to the product of the magnetic moments and inversely proportional to the cube of distance; for two nearly isotropic species, such as quinone radicals, D itself has no significant angular dependence. The most probable absolute value of the angular
Respiratory Electron Transfer Complexes
term is one; orientations for which this is true give rise to the strong pair of inner lines in the spectrum of two dipolar-coupled radicals. As shown by previous workers, the fit of the experimental spectrum to the four lined simulation is impressive. In Fig. 1, the top trace is a simulation of such a four line spectrum caused by the interaction of two isotropic radicals with g= 2.00 and linewidth of 1.2 mT. The value of D was taken as 3.0 mT, which corresponds to a distance slightly less than 0.8 nm. In the following three traces, the simulations depict spectra resulting from the splitting of a radical by a low-spin ferrihaem with g values of 3.4, 1.7 and 0.9, which should be a reasonable approximation of the g-tensor of cytochrome bshOThe traces correspond to orientation of the interspin vector along the maximum g value of the haem, along the minimum g value of the haem, and along the direction equidistant from the principal axes of the g-tensor coordinate system. Owing to the well known ‘3/2’ effect which reduces the effect of dipolar coupling between unlike spins by rendering the off diagonal matrix elements of the dipolar Hamiltonian ineffective, reproducing the splittings approximately would necessitate reducing the spin-spin distance from about 0.77 to about 0.68 nm. The details of the spectrum are not well reproduced for any choice of parameters because the angular dependence of the ferrihaem g-tensor, applied through D,is overlayed on the angular dependence of the dipolar coupling. In Fig. 2, the top trace is a simulation of dipolar coupling between the same ferrihaem and radical with the interspin vector along the direction of the g-tensor co-ordinate system corresponding to the intermediate g value. In this case the outer lines do not correspond to a unique direction, but result from orientations in which either orientation corresponding to the largest or to the intermediate haem g value is along the applied field direction. Spectra of the split signal in oriented multilayers are consistent with dipolar coupling between radicals in which the interspin vector is along the normal to the membrane plane, but would not be consistent with the angular dependence of spectra such as this. The lower trace of Fig. 2 represents the use of a simulation with both isotropic exchange and dipolar coupling between a haem and a radical to obtain a four line pattern with the same spacing as the initial spectrum, which resulted from dipolar coupling alone between two radicals. Exchange interactions could not significantly contribute to the splitting in the case of the two coupled radicals, because they are nearly isotropic and hence can differ in resonant field bv no more than a few tenths __ . I
Fig. I
Computer simulations of e.p.r. spectra of spincoupled pairs Field range is 0.31 t o 0.33 mT and resonant frequency is 9.1 GHz. Parameters for spectra are, in order from top. ( a ) Dipolar-coupled radical spectrum, g values isotropic at 2.0, linewidths I .2 mT, D = 3 mT. ( b ) Dipolar-coupled radical and slowspin ferrihaem, D = 4.5 mT, haem g, = 3.4, g, = I .7, gy= 0.9 with interspin vector along z axis. (c) As above, with interspin vector axis along x axis. ( d ) As above, with interspin vector equidistant from x , y and z axes.
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Magnetic field
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Fig. 2
Computer simulations of haem-radical-coupled pair as in Fig. I.
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Top: interspin vector along y axis. Bottom: with dipolar and exchange terms to produce four line spectrum; interspin vector along z axis, D = 6.6 mT, J = 2.4 mT.
0.01 T
V Magnetic field
of a mT. Note that the outer lines of the upper trace of Fig. 1 are about twice as intense relative to the inner lines as is the case in the lower trace of Fig. 2. The reason for the low intensity of the outer lines is that the additional orientation dependence introduced by the g-tensor anisotropy of the haem is still in effect; the splitting in this case therefore varies much faster with angle than in the dipolar-coupled biradical, and many fewer spins lie near the extrema. It appears to be impossible to simulate the e.p.r. spectra assuming coupling between the haem and a radical. Since both the orientation dependence of the split signal and the redox titration data argue for a ubisemiquinone pair, which accounts well for the observed spectra, it does not appear feasible to substitute a haem for one of the radicals. On the other hand, it is clear that the Qs site is intimately associated with cytochrome bS6@Only SQR or succinate :cytochrome c reductase (SCR) preparations which contain high levels of the
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cytochrome exhibit the split signal. SQR preparations isolated or reconstituted under conditions in which cytochrome bshOis low exhibit a semiquinone radical which relaxes faster than the magnetically isolated Qi radical of the quino1:cytochrome c reductase complex, but is much slower relaxing than the Qs radical in more intact systems [14]. This suggests that the haem is indeed providing part of the relaxation pathway for the radical; the additional rapid relaxation presumably arises from centre S-3. It also appears that the integrity of the Qs site depends on the presence of the haem, which is believed to cross-link the two anchor polypeptides. Yu etal [ 151 showed that the e.p.r. spectrum of the haem reversibly and dramatically shifted upon dissociation of the two soluble subunits. The e.p.r. and optical spectra of the haem are sensitive to the binding of inhibitors which block electron transfer from centre S-3 to quinone; Kd values determined for such inhibitors using the red shift of the optical spectra of cytochrome bshOwhich they produce are comparable with K, values for inhibition of electron transfer [ 141. It is remarkable that the SQR system, which should not, from a chemiosmotic standpoint, require a membrane-inserted electron-carrying arm, has a cytochrome component, while fumarate reductase, which should require transmembrane electron transfer for chemiosmotic function, is devoid of such a component in the E. coli system. In fumarate reductases which have associated cytochromes, Garlands suggestion that they represent a transmembrane electron carrying arm remains attractive. The b haem in the E. coli SQR may have a vestigal structural role; the Qs pair may function to move reducing equivalents and protons into the bilayer. It is possible that the menaquinone binding site in the E. coli QFR system is connected to the surface via a channel, but it is also possible that two or more Q binding sites are present. If this is the case, arrangement of the Q sites to span the membrane, or at least enough of it to bridge the barrier to protonic movement, would increase the Q-Q distance to the point where dipolar coupling would be too weak to produce splittings. This would also render direct proton transfer between quinones unlikely, while electron transfer, which can proceed over large distances, might be catalytically competent. The low potential of centre S-2 has long been a point of contention between workers in the field; for years many laboratories found it difficult to accept the existence of such a low-potential com-
Respiratory Electron Transfer Complexes
ponent in a system reduced by a 0 mV substrate couple. Centre S-2 is not well isolated from other components, however. Interactions between centres S-1 and S-2 have been established for years. Previously, simulations of the splitting of the e.p.r. spectrum of centre s-1 by centre s-2 were performed assuming identical g-tensors but different orientations and suggested that the distance between them was 1-1.2 nm [9a]. Fig. 3 shows similar simulations of the splitting of the central resonance of centre S-2 as in the previously published spectra of reconstitutively inactive preparations of succinate dehydrogenase. Such preparations lack centre S-3, which also interacts with centre S-2 and modulates the S-1/S-2 interaction; they are therefore more easily approached. The top trace shows a simulation in which dipolar coupling consistent with 1 nm separation and 0.2 m T of exchange coupling were Fig. 3 Simulation of splitting of the e.p.r. spectrum of S- I by centre S-2 Field range is 0.30 t o 0.36 mT. Top trace shows dipolar- and exchange coupling of I and 0.2 mT respectively. g values (S-I) 2.03, 1.93, 1.905 (S-2) 2.06, 1.92, 1.85. lnterspin vector oriented 62” (e), 85” Lower trace shows spectral simulation without interaction terms.
(I).
Magnetic field
used; the lower trace shows the simulated spectra of S-1 and S-2 in the absence of coupling. More detailed simulations may bring out additional details, but clearly the distance and orientation with respect to the S-1 cluster g-tensor co-ordinate system deduced from the previous simulations are approximately correct. More recently, it has been proposed that interactions between S-2 and S-3 account for the wings of the e.p.r. spectra in succinate dehydrogenases and fumarate reductases in the fully reduced state. These wings are indicative of much closer coupling between S-2 and S-3 than between S-1 and S-2. Because of the proximity of these centres, it is of dubious validity to treat them as thermodynamically independent entities in analysing the thermodynamics of electron transfer.
Thermodynamics and mechanism The flavin semiquinone in the SQR system is only slightly unstable at pH 7 . Furthermore, both the FADHJFADH and FADH/FAD couples (E”’= - 30 and - 130 mV, respectively) are capable of reducing centre S- 1 without difficulty; the two electron reduction of flavin by succinate is slightly endergonic but can be readily pulled by electron transfer to the higher potential iron-sulphur centres. The uphill two electron step is not helped by division of the following pathway into high- and low-potential arms. The bound semiquinones at the Qs sites associated with SQR are even more stabilized, having at physiological pH stability constants of order unity. These bound quinones are nearly isopotential with centre S-3. The Qs site is thus a highly effective n = l / n = 2 converter well suited to facilitating reduction of ubiquinone to ubiquinol by centre S-3 in two sequential one electron steps. In the QFR system in E. coli, the stability of both the flavosemiquinone radical and a menasemiquinone radical are comparable with the stability of flavosemiquinone in SQR, the two electron potentials for these species are about - 12 mV and - 80 mV respectively (D. Simpkin & W. J. Ingledew, unpublished work). The fumarate reductase system thus is equipped with two potential n = l / n = 2 converters in much the same way as in SQR. As in the SQR system, the low-potential one-electron couples for both flavin and quinone are only 40-50 mV lower in potential than the two-electron couples for each; they are thus more nearly isopotential with the succinate reducible clusters than with the lowpotential centres s-2 or FR-2. If the potentials and stabilities of the radical species associated with QFR and SQR are not well
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suited to a dual pathway scheme for either enzyme, the question of the low potentials of centre S-2 and centre FR-2 remains. Earlier, my co-workers and I pointed out the possibility that interactions between S-1 and S-2 contributed to the low apparent midpoint potential of the latter centre, which might participate in electron transfer by being transiently reduced with an effective potential only 60-100 mV more negative than that of centre S-1 [9a]. Since then it has become clear that S-2 and S-3 are even more closely associated than S-1 and S-2. If we consider the possibility that s-1, s-2 and s-3 are arranged in that order spatially, which is consistent with the strengths of the magnetic interaction between them, we can model the titrations of the three iron-sulphur clusters by assigning interactions between S-1 and S-2 and between S-2 and S-3, and assuming that S-1 and S-3 do not significantly interact directly. We assume that from the oxidized enzyme one-electron reduction of S-3 has a midpoint potential of 60 mV; one-electron reduction of either S-1 or S-2 would have a potential of 0 mV. Interactions between S-2 and S-3 cause the midpoint of either to be lowered by 180 mV upon reduction of the other; an interaction between S-1 and S-2 causes a similar lowering of the mid-point of either by 60 mV upon reduction of the other. In an equilibrium titration, the dominant one-electron form of the enzyme would have centre S-3 reduced; only about 5% of the one-electron reduced enzymes would have centre S-2 reduced; since in these enzymes S-3 would be oxidized and hence odd electron, S-2 would be unlikely to be directly detectable. The dominant two-electron reduced state would have S-1 and S-3 reduced; only 1% of the two-electron reduced enzymes would have S-2 reduced, and in most of these S-3 would also be odd electron. As a result, the titration of S-2 would be observed as an almost ideal n = 1 species at - 240 mV; S-1 and S-3 would titrate as almost ideal n = 1 species at 0 and 60 mV respectively. The behaviour of the iron-sulphur centres in QFR can be as successfully simulated by assigning FR-2 and FR-3 potentials of - 60 mV and FR-1 a potential of 0 mV when all the other centres are oxidized; the interaction terms would be the same as in the SQR case. The result would be nearly ideal n = 1 titrations for FR-1, FR-2 and FR-3 at 0 mV, - 300 mV, and - 60 mV respectively. The potentials which would determine the function of the low-poteniial centres in electron transfer would be mechanism-dependent. Starting from a reduced resting state in which S-1 and S-3 were reduced and S-2 oxidized, reduction of centre
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S-2 by either centre would be unfavourable until S-3 was oxidized by quinol. At that point, the equilibrium constant for electron transfer between S-1 and S-2 would be unity. Once S-2 was reduced, reduction of S-3 would be favoured by a factor of ten. As soon as S-2 was oxidized by S-3, reduction of S- 1 could readily proceed. Starting from the oxidized state, electron transfer to S-1 from flavin would be followed by electron transfer from S-1 to S-2 with an equilibrium constant of unity, after which reduction of S-3 and reduction of S-1 by flavin could proceed. None of these steps need be significantly endergonic. In the same way, reduction of FR-2 by FR-3 in QFR could proceed with a microscopic equilibrium constant of unity even though in equilibrium titration experiments no significant reduction of FR-2 is observed with menaquinol or succinate as reductants. After the oxidation of FR-1 by flavin, FR-2 and FR-3 are essentially isopotential for the exchange of electrons. What are the sources of interactions between electron carriers which contribute to anti-co-operative behaviour? While conformationally-mediated interactions may contribute, electrostatic interactions are strong enough to account for terms of the magnitude needed to account for the observed behaviour. In free space, interactions between single electrons separated by 1 nm give an electrostatic potential energy term of 1.44 eV. Since the S-1 to S-2 distance is approximately 1 nm, an electrostatic term of 60 mV could be produced by a dielectric constant of 24. This is a very high dielectric constant compared with those usually used to represent the interior of proteins; water, with a dielectric constant of 80, is a much more effective quencher of electrostatic interactions. It is likely that the S-2 to S-3 distance is somewhat shorter than 1 nm; the potential due to a point charge is proportional to l/r, so the dielectric constant needed to produce 180 mV of interaction would probably be about 10. Since the two interaction terms could vary considerably (as long as their sum remained constant) and still account for the titration data, a uniform dielectric constant of 12-15 could provide the interactions needed. Given the proximity of the clusters, their location in a protein of substantial size, and the strength of electrostatic interactions over molecular dimensions, the real question is why larger interactions are not observed in proteins with multiple electron carriers. Bulk dielectric constants are not a replacement for the detailed calculations of electrostatic
Respiratory Electron Transfer Complexes
interactions which can be done when more structural information is available, but they do provide order of magnitude estimates of interaction strengths. Evidently, considerable rearrangement of charge driven by reduction significantly raises the effective dielectric. It seems likely that centres S-2 and FR-2 are apparently low-potential clusters merely because they are the middle clusters in the sequence of electron transfer. Interactions in the equilibrium state favour the separation of electrons; at lower levels of reduction, transient electron transfer through the intermediate cluster could be feasible without energetic cost. 1. Ohnishi, T. (1987) Curr. Top. Bioenerg. 15, 37-65 2. Cole, S. T., Condon, C., Lemire, B. D. & Weiner, J. H. (1986) Biochim. Biophys. Acta 135-381 3. Hedersted, I,. & Rutberg, I,. (1981) Microbiol. Kev. 45,542-555 4. Johnson, M. K., Morningstar, J. E., Bennett, D. E., Ackrell, €3. A. C. & Kearney, E. B. (1985) J. Biol. Chem. 260,7368-7378 5. Ohnishi, T., King, T. E., Blum, H., Bowyer, J. R. & Maida, T. (1981)J. Biol. Chem. 256,5577-5582 6. Cammack, R., Crowe, B. A. & Cook, N. D. (1986) Biochem. Soc. Trans. 14, 1207-1208 7. Simpkin, D. & Ingledew, W. J. (1985) Biochem. SOC. Trans. 13,603-607 8. Johnson, M. K., Kowal, A. T., Morningstar, J. E.,
Oliver, M. E., Whittaker, K., Gunsalus, R. P., Ackrell, B. A. C. & Cecchini, G. (1988) J. Biol. Chem. 263, 14732-14738 8a. Johnson, M. K., Morningstar, J. E., Kearney, E. H. & Ackrell, B. A. C. (1988) in Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, B. & Ernster, I,., eds.), pp. 473-483, Plenum Press, N.Y. 9. Salerno, J. C. & Xu, Y. (1988) in Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, €3. & Ernster, I,., eds.), pp. 467-472, Plenum Press, N.Y. 9a. Salerno,J. C., Lim, J., King, T. E., Blum, J. & Ohnishi, T. (1979)J. Biol. Chem. 254,4828-4835 10. Cammack, R., Maguire, J. I. & Ackrell, B. A. C. (1988) in Cytochrome Systems: Molecular Biology and Bioenergetics (Papa, S., Chance, B. & Ernster, I,., eds.), pp. 485-491, Plenum Press, N.Y. 11. Ruzicka, F., Beinert, H., Schepler, K. L., Dunham, W. K. & Sands, K. H. (1975) Proc. Natl. Acad. Sci. U S A . 72,2886 12. Ingledew, W. J., Salerno, J. C. & Ohnishi, T. (1976) Arch. Biochem. Biophys. 177, 176-184 13. Salerno, J. C. & Ohnishi, T. (1980) Biochem. J. 192, 769-78 1 14. Xu, Y., Salerno,J. C.. Wei, Y. H. & King, T. E. (1987) Biochem. Biophys. Res. Commun. 144, 123-128 15. Yu, I,., Xu, J. X., Haley, P. E. & Yu, C. A. (1987) J. Biol. Chem. 262,1137-1 143 Received 18 April 1991
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Bacterial dimethyl sulphoxide reductases and nitrate reductases
Alastair G. McEwan,* Neil Benson,* Tracey C. Bennett,* Steven P. Hanlon,* Stuart J. Fergus0n.t David 1. Richardson,t and J. Baz Jackson$ *School of Biological Sciences, University of East Anglia, Norwich, U.K.,+Department of Biochemistry, University of Oxford, Oxford, U.K. and $School of Biochemistry, University of Birmingham, Birmingham, U.K.
Introduction A wide variety of bacteria can respire using nitrate as an electron acceptor. Nitrate reductase catalyses the two electron reduction of nitrate to nitrite, and this reaction is the first step in denitrification. Nitrate reductases are, however, not restricted to denitrifying bacteria, and many enteric bacteria, for example, Escherichia coli, are capable of nitrate respiration [l]. In addition to their importance in the Nitrogen Cycle, bacteria also have a major role in the cycling of sulphur through their use of oxidized Abbreviations used: DMSO, dimethyl sulphoxide; DMS, dimethyl sulphide; nay, nitrate reductase gene; dms, DMSO reductase gene.
sulphur compounds as electron acceptors. Although the reduction of inorganic sulphur compounds such as sulphate is well established it is now also known that about 50% of the global flux of sulphur is in the form of organic sulphur, especially dimethyl sulphoxide (DMSO) and dimethyl sulphide (DMS) [Z]. DMSO reductase catalyses the two electron reduction of DMSO generating DMS, and over the last decade many bacteria which can respire using DMSO as an electron acceptor have been identified. These include photosynthetic bacteria of the genus Rhodobacter and enteric bacteria exemplified by E. coli A unifying property of nitrate reductases and DMSO reductases is that they are molybdenumcontaining enzymes [ 3 ] .However, there are signifi-
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