A proposed pathway of proton translocation through thebc complexes of mitochondria and chloroplasts

The cytochromebc complexes of the electron transport chain from a wide variety of organisms generate an electrochemical proton gradient which is used for the synthesis of ATP. Proton translocation studies with radiolabeled N,N-dicyclohexylcarbodiimide (DCCD), the well-established carboxyl-modifying reagent, inhibited proton-translocation 50–70% with minimal effect on electron transfer in the cytochromebc ₁ and cytochromebf complexes reconstituted into liposomes. Subsequent binding studies with cytochromebc ₁ and cytochromebf complexes indicate that DCCD specifically binds to the subunitb and subunitb ₆, respectively, in a time and concentration dependent manner. Further analyses of the results with cyanogen bromide and protease digestion suggest that the probable site of DCCD binding is aspartate 160 of yeast cytochromeb and aspartate 155 or glutamate 166 of spinach cytochromeb ₆. Moreover, similar inhibition of proton translocating activity and binding to cytochromeb and cytochromeb ₆ were noticed with N-cyclo-N-(4-dimethylamino-napthyl)carbodiimide (NCD-4), a fluorescent analogue of DCCD. The spin-label quenching experiments provide further evidence that the binding site for NCD-4 on helix cd of both cytochromeb and cytochromeb ₆ is localized near the surface of the membrane but shielded from the external medium.     

Modes of coenzyme Q function in electron transfer

Summary In the mitochondrial respiratory chain, coenzyme Q acts in different ways. A diffusable coenzyme Q pool as a common substrate-like intermediate links the low-potential complexes with complex III. Its diffusion in the lipids is not rate-limiting for electron transfer, but its content is not saturating for maximal rate of NADH oxidation. Protein-bound coenzyme Q is involved in energy conservation, and may be part of enzyme supercomplexes, as in succinate cytochromec reductase. The reason for lack of kinetic saturation of the respiratory chain by quinone concentration is in the low extent of solubility of monomeric coenzyme Q in the membrane lipids. Assays of respiratory enzymes are performed using water soluble coenzyme Q homologs and analogs; several problems exist in using oxidized quinones as acceptors of coenzyme Q reductases. In particular, for complex I no acceptor appears to favorably substitute the endogenous quinone. In addition, quinone reduction sites in complex III compete with the sites in the dehydrogenases, particularly when using duroquinone. The different extent by which these sites operate when different donor substrates (NADH, succinate, glycerol-3-phosphate) are used is best explained by different exposure of the quinone acceptor sites in the dehydrogenases.     

Decoupling of thebc1 complex inS. cerevisiae; point mutations affecting the cytochromeb gene bring new information about the structural aspect of the proton translocation

Four mutations in the mitochondrial cytochromeb ofS. cerevisiae have been characterized with respect to growth capacities, catalytic properties, ATP/2e ^– ratio, and transmembrane potential. The respiratory-deficient mutant G137E and the three pseudo-wild type revertants E137 + I147F, E137 + C133S, and E137 + N256K were described previously (Tron and Lemesle-Meunier, 1990; Di Ragoet al., 1990a). The mutant G137E is unable to grow on respiratory substrates but its electron transfer activity is partly conserved and totally inhibited by antimycin A. The secondary mutations restore the respiratory growth at variable degree, with a phosphorylation efficiency of 12–42% as regards the parental wild type strain, and result in a slight increase in the various electron transfer activities at the level of the whole respiratory chain. The catalytic efficiency for ubiquinol was slightly (G137E) or not affected (E137 + I147F, E137 + C133S, and E137 + N256K) in these mutants. Mutation G137E induces a decrease in the ATP/2e ^– ratio (50% of the W.T. value) and transmembrane potential (60% of the W.T. value) at thebc1 level, whereas the energetic capacity of the cytochrome oxidase is conserved. Secondary mutations I147F, C133S, and N256K partly restore the ATP/ 2e ^– ratio and the transmembrane potential at thebc1 complex level. The results suggest that a partial decoupling of thebc1 complex is induced by the cytochromeb point mutation G137E. In the framework of the protonmotive Q cycle, this decoupling can be explained by the existence of a proton wire connecting centers P and N in the wild typebc1 complex which may be amplified or uncovered by the G137E mutation when the bc1 complex is functioning.     

Electron transfer from cytochromeb 5 to cytochromec

The reaction of cytochromeb ₅ with cytochromec has become a very prominent system for investigating fundamental questions regarding interprotein electron transfer. One of the first computer modeling studies of electron transfer and protein/protein interaction was reported using this system. Subsequently, numerous studies focused on the experimental determination of the features which control protein/protein interactions. Kinetic measurements of the intracomplex electron transfer reaction have only appeared in the last 10 years. The current review will provide a summary of the kinetic measurements and a critical assessment of the interpretation of these experiments.     

Mechanistic and phenomenological features of proton pumps in the respiratory chain of mitochondria

Various direct, indirect (kinetic and thermodynamic), and combined mechanisms have been proposed to explain the conversion of redox energy into a transmembrane protonmotive force (p) by enzymatic complexes of respiratory chains. The conceptual evolution of these models is examined. The characteristics of thermodynamic coupling between redox transitions of electron carriers and scalar proton transfer in cytochromec oxidase and its possible involvement in proton pumping is discussed. Other aspects dealt with in this paper are: (i) variability of H⁺/e ^– stoichiometries, in cytochromec oxidase and cytochromec reductase and its mechanistic implications; (ii) possible models by which the reduction of dioxygen to water at the binuclear heme-copper center of protonmotive oxidases can be directly involved in proton pumping. Finally a unifying concept for proton pumping by the redox complexes of respiratory chain is presented.     

Ascorbate-independent electron transfer between cytochromeb 561 and a 27 kDa ascorbate peroxidase of bean hypocotyls

Summary Cytochromeb ₅₆₁ (cytb ₅₆₁) is a trans-membrane cytochrome probably ubiquitous in plant cells. In vitro, it is readily reduced by ascorbate or by juglonol, which in plasma membrane (PM) preparations from plant tissues is efficiently produced by a PM-associated NAD(P)Hquinone reductase activity. In bean hypocotyl PM, juglonol-reduced cytb ₅₆₁ was not oxidized by hydrogen peroxide alone, but hydrogen peroxide led to complete oxidation of the cytochrome in the presence of a peroxidase found in apoplastic extracts of bean hypocotyls. This peroxidase active on cytb ₅₆₁ was purified from the apoplastic extract and identified as an ascorbate peroxidase of the cytosolic type. The identification was based on several grounds, including the ascorbate peroxidase activity (albeit labile), the apparent molecular mass of the subunit of 27 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the dimeric native structure, the typical spectral properties of a heme-containing peroxidase, and an N-terminal sequence strongly conserved with cytosolic ascorbate peroxidases of plants. Cytb ₅₆₁ used in the experiments was purified from bean hypocotyl PM and juglonol was enzymatically produced by recombinant NAD(P)H:quinone reductase. It is shown that NADPH, NAD(P)H:quinone reductase, juglone, cytb ₅₆₁, the peroxidase interacting with cytb ₅₆₁, and H₂O₂, in this order, constitute an artificial electron transfer chain in which cytb ₅₆₁ is indirectly reduced by NADPH and indirectly oxidized by H₂O₂.Abbreviations APX ascorbate peroxidase - b ₅₆₁PX cytochrome 6561 peroxidase - CPX coniferol peroxidase - cyt cytochrome - GPX guaia-col peroxidase - IWF intercellular washing fluid - MDHA monodehydroascorbate - PM plasma membrane     

A study on electron transport-driven proton translocation in Desulfovibrio desulfuricans

Proton translocation by washed cells of the sulfate-reducing bacterium Desulfovibrio desulfuricans strain Essex 6 was studied by means of pH and sulfide electrodes. Reversible extrusion of protons could be induced either by addition of electron acceptors to cells incubated under hydrogen, or by addition of hydrogen to cells incubated in the presence of an appropriate electron acceptor. Proton translocation was increased in the presence of ionophores that dissipate the membrane potential (thiocyanate, methyl triphenylphosphonium cation, but not valinomycin) and was sensitive to the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). Upon micromolar additions of H₂, usually sulfide was formed in stoichiometric amounts, and extrapolated H⁺/H₂ ratios were 1.8±0.5 with sulfate, 2.3±0.3 with sulfite and 0.5±0.1 with thiosulfate. In several experiments hydrogen pulses caused increased proton extrusion not associated with sulfide production. This was a hint that sulfite might be reduced via intermediates. In the absence of H₂S formation, extrapolated H⁺/H₂ ratios were 3.1±0.8 with sulfate, 3.4±1.1 with sulfite, 4.4±0.8 with thiosulfate and 6.3±1.2 with oxygen. Micromolar pulses of electron acceptors to cells incubated under H₂ caused less proton translocation than H₂ pulses in presence of excess of electron acceptor; extrapolated H⁺/H₂ ratios were 1.3±0.4 with sulfite, 3.3±0.9 with nitrite and 4.2±0.5 with oxygen. No proton translocation was observed after micromolar pulses of sulfate, thiosulfate or nitrate to cells incubated under hydrogen in the presence of thiocyanate. Inhibition experiments with CO and CuCl₂ revealed that the hydrogenase activity was localized in the intracellular space, and that no periplasmic hydrogenase was present. The results indicate that D. desulfuricans can generate a proton gradient by pumping protons across the cytoplasmic membrane.Abbreviations APS adenosine 5-phosphosulfate - CCCP carbonyl cyanide m-chlorophenylhydrazone - MTTP⁺ methyl triphenylphosphonium cation     

Comparative and evolutionary aspects of the photosynthetic electron transfer system of purple bacteria

The cyclic electron transfer system in purple bacteria is composed of the photosynthetic reaction center, the cytochromebc ₁ complex, cytochromec ₂, and ubiquinone. These components share many characteristics with those of photosynthesis and respiration in other organisms. Studies of the cyclic electron transfer system have provided useful insights about the evolution and general mechanisms of membranous energy-conserving systems. The photosynthetic system in purple bacteria may represent a prototype of highly efficient, energy-conserving electron transfer systems in the organisms. Recipient of the Botanical Society Award of Young Scientists, 1992     

Chloroplast-encoded small subunits of the three multiprotein complexes in photosynthetic electron transport

The photosystem I, photosystem II, and cytochromeb ₆ f complexes that are involved in electron transport of oxygenic photosynthesis consist of a number of subunits encoded by either the chloroplast or nuclear genomes. In addition to the major subunits that carry redox components or photosynthetic pigments, these complexes contain several to more than ten subunits with molecular masses of less than 10 kDa. Directed mutagenesis has served as a powerful tool for investigation of the roles of these small subunits in the organization or function of the complexes. Various chloroplast transformants of the green algaChlamydomonas reinhardtii and mutants of cyanobacteria in which a gene encoding a small subunit was deleted or altered have been constructed. Evidence has accumulated suggesting that these small subunits function in the assembly, stabilization, or protection from photoinhibition of the complexes or in the modulation or regulation of electron transport. This article presents an overview of the properties and functions of the chloroplast-encoded small subunits of the three multiprotein complexes of photosynthetic electron transport that have been mainly analyzed with chloroplast transformants ofC. reinhardtii and the corresponding cyanobacterial transformants. Recipient of the Botanical Society Award for Young Scientists, 1995.     

On the role of subunit III in proton translocation in cytochromec oxidase

Mammalian mitochondrial cytochromec oxidase catalyzes the transfer of electrons from ferrocytochromec to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III,M _r 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.     

Photosystem II electron transfer cycle and chlororespiration in planktonic diatoms

The dominance of diatoms in turbulent waters suggests special adaptations to the wide fluctuations in light intensity that phytoplankton must cope with in such an environment. Our recent demonstration of the unusually effective photoprotection by the xanthophyll cycle in diatoms [Lavaud et al. (2002) Plant Physiol 129 (3) (in press)] also revealed that failure of this protection led to inactivation of oxygen evolution, but not to the expected photoinhibition. Photo-oxidative damage might be prevented by an electron transfer cycle around Photosystem II (PS II). The induction of such a cycle at high light intensity was verified by measurements of the flash number dependence of oxygen production in a series of single-turnover flashes. After a few minutes of saturating illumination, the oxygen flash yields are temporarily decreased. The deficit in oxygen production amounts to at most 3 electrons per PS II, but continues to reappear with a half time of 2 min in the dark until the total pool of reducing equivalents accumulated during the illumination has been consumed by (chloro)respiration. This is attributed to an electron transfer pathway from the plastoquinone pool or the acceptor side of PS II to the donor side of PS II that is insignificant at limiting light intensity but is accelerated to milliseconds at excess light intensity. Partial filling of the 3-equivalents capacity of the cyclic electron transfer path in PS II may prevent both acceptor-side photoinhibition in oxygen-evolving PS II and donor-side photoinhibition when the oxygen-evolving complex is temporarily inactivated. This revised version was published online in June 2006 with corrections to the Cover Date.     

A clarification of the effects of DCCD on the electron transfer and antimycin binding of the mitochondrialbc 1 complex

We have studied in detail the effects of dicyclohexylcarbodiimide (DCCD) on the redox activity of the mitochondrialbc ₁ complex, and on the binding of its most specific inhibitor antimycin. An inhibitory action of the reagent has been found only at high concentration of the diimide and/or at prolonged times of incubation. Under these conditions, DCCD also displaced antimycin from its specific binding site in thebc ₁ complex, but did not apparently change the antimycin sensitivity of the ubiquinol-cytochromec reductase activity. On the other hand, using lower DCCD concentrations and/or short times of incubation, i.e., conditions which usually lead to the specific inhibition of the proton-translocating activity of thebc ₁ complex, no inhibitory effect of DCCD could be detected in the ubiquinol-cytochromec reductase activity. However, a clear stimulation of the rate of cytochromeb reduction in parallel to an inhibition of cytochromeb oxidation has been found under these conditions. On the basis of the present work and of previous reports in the literature about the effects of DCCD on thebc ₁ complex, we propose a clarification of the various effects of the reagent depending on the experimental conditions employed.     

The three-subunit cytochromebc 1 complex ofParacoccus denitrificans. Its physiological function,structure, and mechanism of electron transfer and energy transduction

The cytochromebc ₁ complex purified fromP. denitrificans has the same electron-transfer and energy-transducing activities, is sensitive to the same electron-transfer inhibitors, and contains cytochromesb, c ₁, iron-sulfur protein, and thermodynamically stable ubisemiquinone identical to the counterpart complexes from mitochondria. However, the bacterialbc ₁ complex consists of only three proteins, the obligate electron-transfer proteins, while the mitochondrial complexes contain six or more supernumerary poly-peptides, which have no obvious electron-transfer function. TheP. denitrificans complex is a paradigm for thebc ₁ complexes of all gram-negative bacteria. In addition, because of its simple polypeptide composition and apparently minimal damage during isolation, theP. denitrificans bc ₁ complex is an ideal system in which to study structure-function relationships requisite to energy transduction linked to electron transfer.     

Identification of the polypeptides in the cytochromeb 6/f complex from spinach chloroplasts with redox-center-carrying subunits

An improved procedure for the isolation of the cytochromeb ₆/f complex from spinach chloroplasts is reported. With this preparation up to tenfold higher plastoquinol-plastocyanin oxidoreductase activities were observed. Like the complex obtained by our previous procedure, the complex prepared by the modified way consisted of five polypeptides with apparent molecular masses of 34, 33, 23, 20, and 17 kD, which we call Ia, Ib, II, III, and IV, respectively. In addition, one to three small components with molecular masses below 6 kD were now found to be present. These polypeptides can be extracted with acidic acetone. Cytochromef, cytochromeb ₆, and the Rieske Fe-S protein could be purified from the isolated complex and were shown to be represented by subunits Ia + Ib, II, and III, respectively. The heterogeneity of cytochromef is not understood at present. Estimations of the stoichiometry derived from relative staining intensities with Coomassie blue and amido black gave 1:1:1:1 for the subunits Ia + Ib/II/III/IV, which is interesting in of the presence of two cytochromesb ₆ per cytochromef. Cytochromef titrated as a single-electron acceptor with a pH-independent midpoint potential of +339 mV between pH 6.5 and 8.3, while cytochromeb ₆ was heterogeneous. With the assumption of two components present in equal amounts, two one-electron transitions withE _m(1)=–40 mV andE _m(2)=–172 at pH 6.5 were derived. Both midpoint potentials were pH-dependent.Abbreviation Tris tris(hydroxymethyl)aminomethane - SDS sodium dodecylsulfate - SDS-PAGE SDS polyacrylamide gel electrophoresis - MES 2-(N-morpholino)ethanesulfonic acid     

The pathway of electron transfer in the dimeric QH2: Cytochromec oxidoreductase

The experimental data currently available suggest that QH₂: cytochromec oxidoreductase functions according to a Q-cycle type of mechanism. The molecular weight of the enzyme in a natural or artificial phospholipid bilayer or in solution corresponds to that of a dimer. The pre-steady state kinetics of reduction of the prosthetic groups indicate that the enzyme is functionally dimeric. A double Q cycle is proposed, describing the pathway of electron transfer in the dimeric QH₂: cytochromec oxidoreductase. According to this scheme, the two monomeric halves of the enzyme act in a cooperative fashion to complete the catalytic cycle. It is proposed that high-potential cytochromeb-562 and low-potential cytochromeb-562 act cooperatively, viz. as a functional pair, in the antimycin-sensitive reduction of ubiquinone to ubiquinol.     

Reaction center and UQH2:cytc 2 oxidoreductase act as independent enzymes inRps. sphaeroides

Turnover of the ubiquinol oxidizing site of the UQH₂:cyt c₂ oxidoreductase (b/c ₁ complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb ₅₆₁ in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk ₂ of about 3 × 10⁵ M^–1. The reaction shows saturation at high quinol concentrations, with an apparentK _m of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10^–9 cm^–2 sec^–1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb ₅₆₁ occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c ₁ complex. Oxidation of cytb ₅₆₁ through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb ₅₆₁. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theE _h range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol^–1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.     

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb 5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb ₅ performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb ₅ is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.