BBA - Bioenergetics (v.1807, #10)

Allosteric cooperativity in respiratory proteins by Sergio Papa; Maurizio Brunori (1251-1252).

T-quaternary structure of oxy human adult hemoglobin in the presence of two allosteric effectors, L35 and IHP by Kenji Kanaori; Yusuke Tajiri; Antonio Tsuneshige; Izumi Ishigami; Takashi Ogura; Kunihiko Tajima; Saburo Neya; Takashi Yonetani (1253-1261).
The cooperative O2-binding of hemoglobin (Hb) have been assumed to correlate to change in the quaternary structures of Hb: T(deoxy)- and R(oxy)-quaternary structures, having low and high O2-affinities, respectively. Heterotropic allosteric effectors have been shown to interact not only with deoxy- but also oxy-Hbs causing significant reduction in their O2-affinities and the modulation of cooperativity. In the presence of two potent effectors, L35 and inositol hexaphosphate (IHP) at pH 6.6, Hb exhibits extremely low O2-affinities (K T  = 0.0085 mmHg−1 and K R  = 0.011 mmHg−1) and thus a very low cooperativity (K R /K T  = 1.3 and L 0  = 2.4). 1H-NMR spectra of human adult Hb with these two effectors were examined in order to determine the quaternary state of Hb in solution and to clarify the correlation between the O2-affinities and the structural change of Hb caused by the heterotropic effectors. At pH 6.9, 1H-NMR spectrum of deoxy-Hb in the presence of L35 and IHP showed a marker of the T-quaternary structure (the T-marker) at 14 ppm, originated from inter- dimeric α1β2- (or α2β1-) hydrogen-bonds, and hyperfine-shifted (hfs) signals around 15–25 ppm, caused by high-spin heme-Fe(II)s. Upon addition of O2, the hfs signals disappeared, reflecting that the heme-Fe(II)s are ligated with O2, but the T-marker signals still remained, although slightly shifted and broadened, under the partial pressure of O2 (P O2 ) of 760 mmHg. These NMR results accompanying with visible absorption spectroscopy and visible resonance Raman spectroscopy reveal that oxy-Hb in the presence of L35 and IHP below pH 7 takes the ligated T-quaternary structure under the P O2 of 760 mmHg. The L35-concentration dependence of the T-marker in the presence of IHP indicates that there are more than one kind of L35-binding sites in the ligated T-quaternary structure. The stronger binding sites are probably intra-dimeric binding sites between α1G- and β1G-helices, and the other weaker binding site causes the R → T transition without release of O2. The fluctuation of the tertiary structure of Hb seems to be caused by both the structural perturbation of α1β1 (or α2β2) intra-dimeric interface, where the stronger L35-binding sites exist, and by the IHP-binding to the α1α2- (or β1β2-) cavity. The tertiary structural fluctuation induced by the allosteric effectors may contribute to the significant reduction of the O2-affinity of oxy-Hb, which little depends on the quaternary structures. Therefore, the widely held assumptions of the structure-function correlation of Hb — [the deoxy-state] = [the T-quaternary structure] = [the low O 2 -affinity state] and [the oxy-state] = [the R-quaternary structure] = [the high O 2 -affinity state] and the O 2 -affiny of Hb being regulated by the T/R-quaternary structural transition — are no longer sustainable. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► Oxy-hemoglobin having the R-quaternary structure with a high oxygen-affinity is converted to oxy-hemoglobin having the T-quaternary structure with an extremely low oxygen-affinity upon interaction with potent allosteric effectors (inositol hexaphosphate + L35) in solution. ► The quaternary structure and function (the oxygen-affinity, cooperativity, and Bohr effect) of hemoglobin are independently modulated by the competition between the allosteric effects of oxygen and the opposing effects of heterotropic effectors on hemoglobin. ► The ligation state and the quaternary structure of hemoglobin have no direct correlation with the oxygen-affinity or the MWC low-/high- oxygen-affinity functional states.
Keywords: Hemoglobin; Cooperativity; Allostery; NMR; Allosteric effectors; Quaternary structure;

Hemoglobin allostery: Variations on the theme by Andrea Bellelli; Maurizio Brunori (1262-1272).
The two-state allosteric model of Monod, Wyman and Changeux (1965) offers a simple and elegant, yet very powerful and comprehensive, description of the functional behavior of hemoglobin. Although the extensive body of structural and functional information available is by-and-large consistent with this conceptual framework, some discrepancies between theory and experiment have been extensively discussed and considered to demand modifications of the original hypothesis. More recently the role of tertiary structural changes has been re-analyzed leading to extended kinetic models or indicating that powerful heterotropic effectors may be of paramount importance in controlling the function of human hemoglobin. The aim of this review is to analyze, and possibly reconcile, some discrepancies. We always felt that by looking at hemoglobins other than human HbA, the relative role of tertiary and quaternary allosteric effects may be better understood. The model systems illustrated below are the different hemoglobins from trout's blood, since they are characterized by the most striking variability of heterotropic effects, ranging from totally absent to very extreme with dominant contributions of tertiary effects. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► Allostery demands the coupling between tertiary and quaternary structural changes. ►Hemoglobin is the paradigm of structure–function correlations known in depth. ►Fish hemoglobins display the largest possible variations in allosteric control. ►The concept of allosteric regulation has been extended to monomeric proteins.
Keywords: Two state model; Cooperativity; Tertiary/quaternary structures;

The D-channel of cytochrome oxidase: An alternative view by Mårten Wikström; Michael I. Verkhovsky (1273-1278).
The D-pathway in A-type cytochrome c oxidases conducts protons from a conserved aspartate on the negatively charged N-side of the membrane to a conserved glutamic acid at about the middle of the membrane dielectric. Extensive work in the past has indicated that all four protons pumped across the membrane on reduction of O2 to water are transferred via the D-pathway, and that it is also responsible for transfer of two out of the four “chemical protons” from the N-side to the binuclear oxygen reduction site to form product water. The function of the D-pathway has been discussed in terms of an apparent pK a of the glutamic acid. After reacting fully reduced enzyme with O2, the rate of formation of the F state of the binuclear heme-copper active site was found to be independent of pH up to pH~9, but to drop off at higher pH with an apparent pK a of 9.4, which was attributed to the glutamic acid. Here, we present an alternative view, according to which the pH-dependence is controlled by proton transfer into the aspartate residue at the N-side orifice of the D-pathway. We summarise experimental evidence that favours a proton pump mechanism in which the proton to be pumped is transferred from the glutamic acid to a proton-loading site prior to proton transfer for completion of oxygen reduction chemistry. The mechanism is discussed by which the proton-pumping activity is decoupled from electron transfer by structural alterations of the D-pathway. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► We present a critical analysis of the properties of the so-called D-pathway of proton transfer in the A-type heme-copper oxidases. ► The role of the D-pathway in proton translocation is analysed in terms of side chain isomerisation of the residue Glu286. ► The rate of proton transfer in the D-pathway depends on the product of the rate constant (k)that may be changed by mutations, and the occupancy of the protonated form of Asp132 at the channel orifice.
Keywords: Proton transfer; Proton pumping; Cell respiration; Energy transduction;

The O2 reduction and proton pumping gate mechanism of bovine heart cytochrome c oxidase by Shinya Yoshikawa; Kazumasa Muramoto; Kyoko Shinzawa-Itoh (1279-1286).
X-ray structures of bovine heart cytochrome c oxidase with bound respiratory inhibitors (O2 analogues) have been determined at 1.8–2.05 Å resolution to investigate the function of the O2 reduction site which includes two metal sites (Fe a3 2+ and CuB 1+). The X-ray structures of the CO- and NO-bound derivatives indicate that although there are three possible electron donors that can provide electrons to the bound O2, located in the O2 reduction site, the formation of the peroxide intermediate is effectively prevented to provide an O2-bound form as the initial intermediate. The structural change induced upon binding of CN suggests a non-sequential 3-electoron reduction of the bound O2 for the complete reduction without release of any reactive oxygen species. The X-ray structure of the derivative with CO bound to CuB 1+ after photolysis from Fe a3 2+ demonstrates weak side-on binding. This suggests that CuB controls the O2 supply to Fe a3 2+ without electron transfer to provide sufficient time for collection of protons from the negative side of the mitochondrial membrane. The proton-pumping pathway of bovine heart cytochrome c oxidase includes a hydrogen-bond network and a water channel located in tandem between the positive and negative side of the mitochondrial membrane. Binding of a strong ligand to Fe a3 induces a conformational change which significantly narrows the water channel and effectively blocks the back-leakage of protons from the hydrogen bond network. The proton pumping mechanism proposed by these X-ray structural analyses has been functionally confirmed by mutagenesis analyses of bovine heart cytochrome c oxidase. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► Respiratory inhibitors as probes for mechanism of cytochrome c oxidase function. ► O2 reduction by O2 binding to Fe2+, followed by non-sequential 3-electron reduction ► Positive charges of low spin heme, upon oxidation, pump protons electrostatically. ► X-ray and mutational identification of proton pump system of cytochrome c oxidase.
Keywords: X-ray structural analysis; Membrane protein; Hemoprotein; Terminal oxidase; O2 reduction; Proton pump;

Redox Bohr effects and the role of heme a in the proton pump of bovine heart cytochrome c oxidase by Giuseppe Capitanio; Pietro Luca Martino; Nazzareno Capitanio; Sergio Papa (1287-1294).
Structural and functional observations are reviewed which provide evidence for a central role of redox Bohr effect linked to the low-spin heme a in the proton pump of bovine heart cytochrome c oxidase. Data on the membrane sidedness of Bohr protons linked to anaerobic oxido-reduction of the individual metal centers in the liposome reconstituted oxidase are analysed. Redox Bohr protons coupled to anaerobic oxido-reduction of heme a (and CuA) and CuB exhibit membrane vectoriality, i.e. protons are taken up from the inner space upon reduction of these centers and released in the outer space upon their oxidation. Redox Bohr protons coupled to anaerobic oxido-reduction of heme a 3 do not, on the contrary, exhibit vectorial nature: protons are exchanged only with the outer space. A model of the proton pump of the oxidase, in which redox Bohr protons linked to the low-spin heme a play a central role, is described. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.Display Omitted► In bovine cytochrome c oxidase redox transitions of metal centers result in Bohr H+ effect. ► Redox Bohr H+ coupled to heme a (and CuA) exhibit membrane vectoriality. ► Heme a plays a central role in the proton pump of COX.
Keywords: Cytochrome c oxidase; Redox Bohr effect; Cooperative coupling; Proton pump; Membrane vectoriality;

Radical formation in cytochrome c oxidase by Michelle A. Yu; Tsuyoshi Egawa; Kyoko Shinzawa-Itoh; Shinya Yoshikawa; Syun-Ru Yeh; Denis L. Rousseau; Gary J. Gerfen (1295-1304).
The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O2 redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O2 with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77 K at reaction times ranging from 50 μs to 6 ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9 GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130 GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO2 ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O2 with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H2O2) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50 μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (R―O―O•) species was formed, presumably by the reaction between O2 and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a 3. This article is part of a Special Issue entitled: “Allosteric cooperativity in respiratory proteins”.► Spurious radicals are generated by ascorbate and dithionite. ► Radicals are formed on Y244 and Y129 in the reaction with hydrogen peroxide. ► Peroxyl radicals are formed at 50 μs. ► Y129 may be the proton loading site for proton translocation.
Keywords: Bioenergetics; Electron paramagnetic resonance; Radicals; Proton translocation; Peroxyl;

Electron transfer pathways in cytochrome c oxidase by M. Fátima Lucas; Denis L. Rousseau; Victor Guallar (1305-1313).
Mixed quantum mechanical/molecular mechanics calculations were used to explore the electron pathway of the terminal electron transfer enzyme, cytochrome c oxidase. This enzyme catalyzes the reduction of molecular oxygen to water in a multiple step process. Density functional calculations on the three redox centers allowed for the characterization of the electron transfer mechanism, following the sequence CuA  → heme a  → heme a 3. This process is largely affected by the presence of positive charges, confirming the possibility of a proton coupled electron transfer. An extensive mapping of all residues involved in the electron transfer, between the CuA center (donor) and the O2 reduction site heme a 3-CuB (receptor), was obtained by selectively activating/deactivating different quantum regions. The method employed, called QM/MM e-pathway, allowed the identification of key residues along the possible electron transfer paths, consistent with experimental data. In particular, the role of arginines 481 and 482 appears crucial in the CuA  → heme a and in the heme a  → heme a 3 electron transfer processes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.Display Omitted► Cytochrome c oxidase electron transfer pathways studied by a QM/MM approach. ► Key residues for electron transfer identified. ► Plasticity for electron flow regulated by the neighboring arginines 481 and 482. ► Two step ET process following the CuA -> heme a -> heme a 3 sequence.
Keywords: Cytochrome c oxidase; Electron transfer; QM/MM e-pathway; heme; Bioenergetics;

The functional roles of the amino acid residues of the CuA site in bovine cytochrome c oxidase (CcO) were investigated by utilizing hybrid quantum mechanics (QM)/molecular mechanics (MM) calculations. The energy levels of the molecular orbitals (MOs) involving Cu d zx orbitals unexpectedly increased, as compared with those found previously with a simplified model system lacking the axial Met residue (i.e., Cu2S2N2). This elevation of MO energies stemmed from the formation of the anti-bonding orbitals, which are generated by hybridization between the d zx orbitals of Cu ions and the p-orbitals of the S and O atoms of the axial ligands. To clarify the roles of the axial Met ligand, the inner-sphere reorganization energies of the CuA site were computed, with the Met residue assigned to either the QM or MM region. The reorganization energy slightly increased when the Met residue was excluded from the QM region. The existing experimental data and the present structural modeling study also suggested that the axial Met residue moderately increased the redox potential of the CuA site. Thus, the role of the Met may be to regulate the electron transfer rate through the fine modulation of the electronic structure of the CuA “platform”, created by two Cys/His residues coordinated to the Cu ions. This regulation would provide the optimum redox potential/reorganization energy of the CuA site, and thereby facilitate the subsequent cooperative reactions, such as the proton pump and the enzymatic activity, of CcO. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.Display Omitted► Theoretical study of the CuA site of bovine cytochrome c oxidase. ► Fine-modulation of the electronic state of the CuA “platform” by the axial Met. ► CuA established by this modulation triggers the subsequent cooperative reactions.
Keywords: Transition metal binding protein; Redox potential; Electron transfer; Electronic structure; Structural modeling; Hybrid QM/MM calculation;

We report first-principles molecular dynamics calculations based on density functional theory performed on the entrance part of the D-path pathway in bovine cytochrome c oxidase. Our models, which are extracted from the fully reduced and oxidized X-ray structures, include His503 as a protonatable site. We find that the protonated His503 with the deprotonated Asp91 [H503–Nδ1H+ and D91–CγOOγ] are more energetically favorable than other protonation states, [H503–Nδ1 and D91–CγOOH], with an energy difference of about − 5 kcal/mol in reduced case, while the [H503–Nδ1H+ and D91–CγOO] state is energetically unstable, about + 3 kcal/mol higher in energy in the oxidized case. The local interaction of His503 with the surrounding polar residues is necessary and sufficient for determining the energetics. The redox-coupled rotation of His503 is found to change the energetics of the protonation states. We also find that this rotation is coupled with the proton transfer from His503 and Asp91, which leads to the transition between the two different protonation states. This study suggests that His503 is involved in the proton supply to the D-path as a proton acceptor and that the redox-controlled proton-transfer-coupled rotation of His503 is a key process for an effective proton supply to the D-path from water bulk. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► Protonation states of a histidine located in the entrance of a proton transfer path. ► The energetics is changed by the redox-coupled rotation of the histidine. ► The rotation of the histidine is also coupled with local proton transfer.
Keywords: Cytochrome c oxidase; Histidine; Redox-controlled proton-transfer-coupled rotation; First-principles molecular dynamics; Density functional theory; Metadynamics;

Functional effects of mutations in cytochrome c oxidase related to prostate cancer by Ida Namslauer; Marina S. Dietz; Peter Brzezinski (1336-1341).
A number of missense mutations in subunit I of cytochrome c oxidase (CytcO) have previously been linked to prostate cancer (Petros et al., 2005). To investigate the effects of these mutations at the molecular level, in the present study we prepared four different structural variants of the bacterial Rhodobacter sphaeroides CytcO (cytochrome aa 3), each carrying one amino-acid residue replacement corresponding to the following substitutions identified in the above-mentioned study: Asn11Ser, Ala122Thr, Ala341Ser and Val380Ile (residues Asn25, Ser168, Ala384 and Val423 in the R. sphaeroides oxidase). This bacterial CytcO displays essentially the same structural and functional characteristics as those of the mitochondrial counterpart. We investigated the overall activity, proton pumping and internal electron- and proton-transfer reactions in the structural variants. The results show that the turnover activities of the mutant CytcOs were reduced by at most a factor of two. All variants pumped protons, but in Ser168Thr, Ala384Ser and Val423Ile we observed slight internal proton leaks. In all structural variants the internal electron equilibrium was slightly shifted away from the catalytic site at high pH (10), resulting in a slower observed ferryl to oxidized transition. Even though the effects of the mutations were relatively modest, the results suggest that they destabilize the proton-gating machinery. Such effects could be manifested in the presence of a transmembrane electrochemical gradient resulting in less efficient energy conservation. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► Effects of four mutations identified in prostate cancer patients were characterized at a molecular-functional level. ► The data indicate that the mutations destabilize the enzyme structure thereby influencing proton gating. ► The bacterial R. sphaeroides cytochrome c oxidase was used as a model to investigate effects of disease-related mutations in the enzyme.
Keywords: Respiration; Proton pumping; Electron transfer; Cytochrome aa 3; Mitochondria; Membrane protein;

Differential effects of glutamate-286 mutations in the aa 3-type cytochrome c oxidase from Rhodobacter sphaeroides and the cytochrome bo 3 ubiquinol oxidase from Escherichia coli by Tsuyoshi Egawa; Krithika Ganesan; Myat T. Lin; Michelle A. Yu; Jonathan P. Hosler; Syun-Ru Yeh; Denis L. Rousseau; Robert B. Gennis (1342-1348).
Both the aa 3-type cytochrome c oxidase from Rhodobacter sphaeroides (RsCcO aa3) and the closely related bo 3-type ubiquinol oxidase from Escherichia coli (EcQO bo3) possess a proton-conducting D-channel that terminates at a glutamic acid, E286, which is critical for controlling proton transfer to the active site for oxygen chemistry and to a proton loading site for proton pumping. E286 mutations in each enzyme block proton flux and, therefore, inhibit oxidase function. In the current work, resonance Raman spectroscopy was used to show that the E286A and E286C mutations in RsCcO aa3 result in long range conformational changes that influence the protein interactions with both heme a and heme a 3 . Therefore, the severe reduction of the steady-state activity of the E286 mutants in RsCcO aa3 to ~ 0.05% is not simply a result of the direct blockage of the D-channel, but it is also a consequence of the conformational changes induced by the mutations to heme a and to the heme a 3-CuB active site. In contrast, the E286C mutation of EcQO bo3 exhibits no evidence of conformational changes at the two heme sites, indicating that its reduced activity (3%) is exclusively a result of the inhibition of proton transfer from the D-channel. We propose that in RsCcO aa3, the E286 mutations severely perturb the active site through a close interaction with F282, which lies between E286 and the heme-copper active site. The local structure around E286 in EcQO bo3 is different, providing a rationale for the very different effects of E286 mutations in the two enzymes. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.Display Omitted► We compare E286 mutations in the oxidases from R. sphaeroides and E. coli. ► E286C in E. coli cyt bo3 blocks proton flux through the D-channel. ► E286C/A/Q causes extensive conformational changes in the R. sphaeroides oxidase. ► Differences are ascribed to residues surrounding E286 in the two oxidases.
Keywords: Raman scattering; Bioenergetics; Proton translocation; Mutant; Heme; Copper;

The modified Q cycle mechanism accounts for the proton and charge translocation stoichiometry of the bc 1 complex, and is now widely accepted. However the mechanism by which the requisite bifurcation of electron flow at the Qo site reaction is enforced is not clear. One of several proposals involves conformational gating of the docking of the Rieske ISP at the Qo site, controlled by the stage of the reaction cycle. Effects of different Qo-site inhibitors on the position of the ISP seen in crystals may reflect the same conformational mechanism, in which case understanding how different inhibitors control the position of the ISP may be a key to understanding the enforcement of bifurcation at the Qo site (). Here we examine the available structures of cytochrome bc 1 with different Qo-site inhibitors and different ISP positions to look for clues to this mechanism. The effect of ISP removal on binding affinity of the inhibitors stigmatellin and famoxadone suggest a “mutual stabilization” of inhibitor binding and ISP docking, however this thermodynamic observation sheds little light on the mechanism. The cd1 helix of cytochrome b moves in such a way as to accommodate docking when inhibitors favoring docking are bound, but it is impossible with the current structures to say whether this movement of α-cd1 is a cause or result of ISP docking. One component of the movement of the linker between E and F helices also correlates with the type of inhibitor and ISP position, and seems to be related to the H-bonding pattern of Y279 of cytochrome b. An H-bond from Y279 to the ISP, and its possible modulation by movement of F275 in the presence of famoxadone and related inhibitors, or its competition with an alternate H-bond to I269 of cytochrome b that may be destabilized by bound famoxadone, suggest other possible mechanisms. This article is part of a Special Issue entitled: Allosteric cooperativity in respiratory proteins.► In Cytochrome bc1 , inhibitor-binding affects position of the Iron–Sulfur Protein. ► Inhibitors Stigmatellin and nHDBT "fix" ISP position with cluster near cyt. b. ► Stigmatellin and nHDBT make an H-bond to the ISP which may be responsible. ► New class of inhibitors fix the ISP in a different position without H-bond. ► Mechanism by which inhibitors position ISP may explain strict bifurcation at Qo.
Keywords: Cytochrome bc1; Inhibitor binding; Conformational change; Stigmatellin; Famoxadone; Fenamidone;

Despite its central function in oxidative phosphorylation, the molecular mechanism of proton pumping respiratory complex I is still elusive. In recent years, considerable progress has been made towards understanding structure/function relationships in this very large and complicated membrane protein complex. Last year X-ray crystallographic analysis of bacterial and mitochondrial complex I provided important insights into its molecular architecture. Based on this evidence, here a hypothetical molecular mechanism for redox-driven proton pumping of complex I is proposed. According to this mechanism, two pump modules are driven by two conformational strokes that are generated by stabilization of the anionic forms of semiquinone and ubiquinol that are formed in the peripheral arm of complex I during turnover. This results in the experimentally determined pumping stoichiometry of 4 H+/2e. In the two-state model, electron transfer from iron–sulfur cluster N2 is allowed only in the ‘E-state,’ while protonation of the substrate is only possible in the stabilizing ‘P-state.’ In the membrane arm, transition from the E- to the P-state drives the two pump modules via long range conformational energy transfer through the recently discovered helical transmission element connecting them. The proposed two-state stabilization-change mechanism is fully reversible and thus inherently explains the operation of complex I in forward and reverse mode. This article is part of a Special Issue entitled Allosteric cooperativity in respiratory proteins.Display Omitted► A conformational two-state mechanism for proton pumping complex I is proposed. ►The mechanism relies on stabilization changes of anionic ubiquinone intermediates. ►Electron-transfer and protonation should be strictly controlled during turnover. ►The mechanism explains the full reversibility of complex I.
Keywords: Complex I; Mitochondria; Proton pumping; Mechanism; Ubiquinone;