Inter-monomer electron transfer is too slow to compete with monomeric turnover in bc1 complex

The homodimeric bc₁ complexes are membrane proteins essential in respiration and photosynthesis. The ~ 11 Å distance between the two b_L-hemes of the dimer opens the possibility of electron transfer between them, but contradictory reports make such inter-monomer electron transfer controversial. We have constructed in Rhodobacter sphaeroides a heterodimeric expression system similar to those used before, in which the bc₁ complex can be mutated differentially in the two copies of cyt b to test for inter-monomer electron transfer, but found that genetic recombination by cross-over then occurs to produce wild-type homodimer. Selection pressure under photosynthetic growth always favored the homodimer over heterodimeric variants enforcing inter-monomer electron transfer, showing that the latter are not competitive. These results, together with kinetic analysis of myxothiazol titrations, demonstrate that inter-monomer electron transfer does not occur at rates competitive with monomeric turnover. We examine the results from other groups interpreted as demonstrating rapid inter-monomer electron transfer, conclude that similar mechanisms are likely to be in play, and suggest that such claims might need to be re-examined.     

Hybrid fusions show that inter-monomer electron transfer robustly supports cytochrome bc1 function in vivo

Electronic connection between Q_o and Q_i quinone catalytic sites of dimeric cytochrome bc₁ is a central feature of the energy-conserving Q cycle. While both the intra- and inter-monomer electron transfers were shown to connect the sites in the enzyme, mechanistic and physiological significance of the latter remains unclear. Here, using a series of mutated hybrid cytochrome bc₁-like complexes, we show that inter-monomer electron transfer robustly sustains the function of the enzyme in vivo, even when the two subunits in a dimer come from different species. This indicates that minimal requirement for bioenergetic efficiency is to provide a chain of cofactors for uncompromised electron flux between the catalytic sites, while the details of protein scaffold are secondary.     

The Q-cycle reviewed: How well does a monomeric mechanism of the bc1 complex account for the function of a dimeric complex?

Recent progress in understanding the Q-cycle mechanism of the bc₁ complex is reviewed. The data strongly support a mechanism in which the Q_o-site operates through a reaction in which the first electron transfer from ubiquinol to the oxidized iron–sulfur protein is the rate-determining step for the overall process. The reaction involves a proton-coupled electron transfer down a hydrogen bond between the ubiquinol and a histidine ligand of the [2Fe–2S] cluster, in which the unfavorable protonic configuration contributes a substantial part of the activation barrier. The reaction is endergonic, and the products are an unstable ubisemiquinone at the Q_o-site, and the reduced iron–sulfur protein, the extrinsic mobile domain of which is now free to dissociate and move away from the site to deliver an electron to cyt c₁ and liberate the H⁺. When oxidation of the semiquinone is prevented, it participates in bypass reactions, including superoxide generation if O₂ is available. When the b-heme chain is available as an acceptor, the semiquinone is oxidized in a process in which the proton is passed to the glutamate of the conserved -PEWY- sequence, and the semiquinone anion passes its electron to heme b_L to form the product ubiquinone. The rate is rapid compared to the limiting reaction, and would require movement of the semiquinone closer to heme b_L to enhance the rate constant. The acceptor reactions at the Q_i-site are still controversial, but likely involve a “two-electron gate” in which a stable semiquinone stores an electron. Possible mechanisms to explain the cyt b₁₅₀ phenomenon are discussed, and the information from pulsed-EPR studies about the structure of the intermediate state is reviewed.The mechanism discussed is applicable to a monomeric bc₁ complex. We discuss evidence in the literature that has been interpreted as shown that the dimeric structure participates in a more complicated mechanism involving electron transfer across the dimer interface. We show from myxothiazol titrations and mutational analysis of Tyr-199, which is at the interface between monomers, that no such inter-monomer electron transfer is detected at the level of the b_L hemes. We show from analysis of strains with mutations at Asn-221 that there are coulombic interactions between the b-hemes in a monomer. The data can also be interpreted as showing similar coulombic interaction across the dimer interface, and we discuss mechanistic implications.     

The effect of pre-reduction of cytochrome b-563 on the electron-transfer reactions of the cytochrome bf complex in higher plant chloroplasts

An investigation has been made of the effects of pre-reduction of cytochrome b-563 on electron transfers through the cytochrome bf complex. It has been found that in a system in which anthraquinone-2-sulphonate or anthraquinone-2,6-disulphonate is used as redox buffer, a lipid-soluble mediator must also be present to allow sufficiently rapid equilibration of cytochrome b-563 with the ambient potential. We have found that 1 μM benzyl viologen gives full equilibration of cytochrome b-563 in less than 30 s, while minimizing the side reactions that have been observed with alternative mediators. Pre-reduction of cytochrome b-563 did not prevent turnover of site o (quinol-oxidising site of the cytochrome bc complex), even with fast repetitive flash activation. The site o reaction was accompanied by rapid, 2-nonyl-4-hydroxyquinoline N-oxide-sensitive oxidation of cytochrome b, and by a slow carotenoid bandshift. These results are discussed in conjunction with related results from the cytochrome bc₁ complex; Q-cycle models are considered in which the semiquinone at site o either can reduce an oxidant other than cytochrome b-563, or can migrate to site r (quinone-reducing site of the cytochrome bc complex). Of these possibilities, only the migration of the neutral semiquinone, QH, to site r is compatible with all of the data from the cytochrome bf and bc₁ complexes. Such a scheme would not be compatible with the semiquinone cycle proposed by Wikström and Krab ((1986) J. Bioenerg. Biomembr. 18, 181–193).     

Calcium-dependent release of arachidonic acid from a murine epidermal cell line induced by the tumor promoter TPA or ionophore a 23187: Dissection of ionophoretic and phospholipase A2-stimulating activity

Beef heart mitochondrial bc₁ complex (ubiquinone—cytochrome c oxidoreductase) has been assayed by its ability to catalyse the reaction of duroquinol reduction of ferricytochrome c. When the isolated complex is reincorporated into lipid vesicles, the enzyme-catalysed electron transfer rate becomes uncoupler-sensitive. Initial experiments suggest that a protonmotive force is generated across the vesicles when electron transfer is initiated. Both ΔΨ and ΔpH components of this protonmotive force then influence an internal rate constant of the bc₁ complex.     

Regulatory interactions in the dimeric cytochrome bc1 complex: The advantages of being a twin

The dimeric cytochrome bc₁ complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc₁ complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.     

Oxygen dependent electron transfer in the cytochrome bc1 complex

The effect of molecular oxygen on the electron transfer activity of the cytochrome bc₁ complex was investigated by determining the activity of the complex under the aerobic and anaerobic conditions. Molecular oxygen increases the activity of Rhodobacter sphaeroides bc₁ complex up to 82%, depending on the intactness of the complex. Since oxygen enhances the reduction rate of heme b_L, but shows no effect on the reduction rate of heme b_H, the effect of oxygen in the electron transfer sequence of the cytochrome bc₁ complex is at the step of heme b_L reduction during bifurcated oxidation of ubiquinol.     

FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical ‘chromatophore’ vesicles. These bacterial ‘organelles’ are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc₁ complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c₂. Proximity between cytochrome bc₁ and RC-LH1-PufX minimises quinone/quinol/cytochrome c₂ diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc₁ with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc₁ complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc₁ observed by spectroscopy, and explain how quinols/quinones and cytochrome c₂ shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.     

Enzymatic activity of the alternative complex III as a menaquinol:auracyanin oxidoreductase in the electron transfer chain of Chloroflexus aurantiacus

The surprising lack of the cytochrome bc₁ complex in the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus suggests that a functional replacement exists to link the cyclic electron transfer chain. Earlier work identified the alternative complex III (ACIII) as a substitute of cytochrome bc₁ complex. Herein, the enzymatic activity of ACIII is studied. The results strongly support the view that the ACIII functions as menaquinol:auracyanin oxidoreductase in the C. aurantiacus electron transfer chain. Among all the substrates tested, auracyanin is the most efficient electron acceptor of ACIII, suggesting that ACIII directly transfers the electron to auracyanin instead of cytochrome c-554. The lack of sensitivity to common inhibitors of the cytochrome bc₁ complex indicates a different catalytic mechanism for the ACIII complex.     

The site of inhibition of mitochondrial electron transfer by coenzyme Q analogues.

The effects of 33 quinone derivatives on mitochondrial electron transfer in yeast were examined. Twenty-two of the compounds were also tested for their effects on the growth of yeast cells. Four strong inhibitors of electron transfer were identified: 5-n-undecyl-6-hydroxy-4, 7-dioxobenzothiazole, 7-ω-cyclohexyloctyl-6-hydroxy-5,8-quinolinequinone, 7-n-hexadecyl-mercapto-6-hydroxy-5, 8-quinolinequinone, and 3-n-dodecylmercapto-2-hydroxy-1, 4-naphthoquinone. They inhibit the growth of yeast with ethanol as an energy source, but not when glucose is the energy source. The NADH oxidase activity of isolated mitochondria is 50% inhibited by these quinone derivatives at about 10^?8m, or 0.5 μmol/g mitochondrial protein; 1000-fold higher concentrations do not affect electron transfer from NADH or succinate to coenzyme Q₂. The effects of the inhibitors on cytochrome spectra indicate that they block electron transfer between cytochromes b and c₁. A possible antagonism between these compounds and coenzyme Q at a site between cytochromes b and C₁ is discussed in terms of Mitchell's “protonmotive Q cycle” hypothesis (Mitchell, P. (1976) J. Theor. Biol. 62, 327–367). 6-β-naphthylmercapto-5-chloro-2,3-dimethoxy-1,4-benzoquinone inhibits electron transfer between succinate and coenzyme Q₂ or phenazine methosulfate, suggesting a site in the succinate-coenzyme Q reductase complex with a different quinone specificity from that of the site in the cytochrome bc₁ complex. Seven of the quinone derivatives inhibit growth on both glucose and ethanol media, indicating that their effect is not the result of inhibition of respiration.     

A four-subunit cytochrome bc1 complex complements the respiratory chain of Thermus thermophilus

Several components of the respiratory chain of the eubacterium Thermus thermophilus have previously been characterized to various extent, while no conclusive evidence for a cytochrome bc₁ complex has been obtained. Here, we show that four consecutive genes encoding cytochrome bc₁ subunits are organized in an operon-like structure termed fbcCXFB. The four gene products are identified as genuine subunits of a cytochrome bc₁ complex isolated from membranes of T. thermophilus. While both the cytochrome b and the FeS subunit show typical features of canonical subunits of this respiratory complex, a further membrane-integral component (FbcX) of so far unknown function copurifies as a subunit of this complex. The cytochrome c₁ carries an extensive N-terminal hydrophilic domain, followed by a hydrophobic, presumably membrane-embedded helical region and a typical heme c binding domain. This latter sequence has been expressed in Escherichia coli, and in vitro shown to be a kinetically competent electron donor to cytochrome c₅₅₂, mediating electron transfer to the ba₃ oxidase. Identification of this cytochrome bc₁ complex bridges the gap between the previously reported NADH oxidation activities and terminal oxidases, thus, defining all components of a minimal, mitochondrial-type electron transfer chain in this evolutionary ancient thermophile.     

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc₁) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe₂S₂ cluster, cyt c₁ and cyt b, forming two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the electron transfer pathways originating from QH₂ oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc₁ and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q_o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc₁ activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc₁. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH₂ oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe₂S₂ cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH₂ oxidation at the Q_o site of cyt bc₁.     

Tuning of Hemes b Equilibrium Redox Potential Is Not Required for Cross-Membrane Electron Transfer

In biological energy conversion, cross-membrane electron transfer often involves an assembly of two hemes b. The hemes display a large difference in redox midpoint potentials (ΔE_{m_}b), which in several proteins is assumed to facilitate cross-membrane electron transfer and overcome a barrier of membrane potential. Here we challenge this assumption reporting on heme b ligand mutants of cytochrome bc₁ in which, for the first time in transmembrane cytochrome, one natural histidine has been replaced by lysine without loss of the native low spin type of heme iron. With these mutants we show that ΔE_{m_}b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔE_{m_}b can be markedly decreased to the point that two hemes are almost isopotential, yet the enzyme retains catalytically competent electron transfer between quinone binding sites and remains functional in vivo. This reveals that cytochrome bc₁ can accommodate large changes in ΔE_{m_}b without hampering catalysis, as long as these changes do not impose overly endergonic steps on downhill electron transfer from substrate to product. We propose that hemes b in this cytochrome and in other membranous cytochromes b act as electronic connectors for the catalytic sites with no fine tuning in ΔE_{m_}b required for efficient cross-membrane electron transfer. We link this concept with a natural flexibility in occurrence of several thermodynamic configurations of the direction of electron flow and the direction of the gradient of potential in relation to the vector of the electric membrane potential.     

Effect of Mutations of Arginine 94 on Proton Pumping,Electron Transfer,and Superoxide Anion Generation in Cytochrome b of the bc1 Complex from Rhodobacter sphaeroides

Proton transfer involving internal water molecules that provide hydrogen bonds and facilitate proton diffusion has been identified in some membrane proteins. Arg-94 in cytochrome b of the Rhodobacter sphaeroides bc₁ complex is fully conserved and is hydrogen-bonded to the heme propionate and a chain of water molecules. To further elucidate the role of Arg-94, we generated the mutations R94A, R94D, and R94N. The wild-type and mutant bc₁ complexes were purified and then characterized. The results show that substitution of Arg-94 decreased electron transfer activity and proton pumping capability and increased O₂^˙̄ production, suggesting the importance of Arg-94 in the catalytic mechanism of the bc₁ complex in R. sphaeroides. This also suggests that the transport of H⁺, O₂, and O₂^˙̄ in the bc₁ complex may occur by the same pathway.     

Interaction of cytochrome c with cytochrome bc1 complex of the mitochondrial respiratory chain

The binding of cytochrome c to the cytochrome bc₁ complex of bovine heart mitochondria was studied. Cytochrome c derivatives, arylazido-labeled at lysine 13 or lysine 22, were prepared and their properties as electron acceptors from the bc₁ complex were measured. Mixtures of bc₁ complex with cytochrome c derivatives were illuminated with ultraviolet light and afterwards subjected to polyacrylamide gel electrophoresis. The gels were analysed using dualwavelength scanning at 280 minus 300 and 400 minus 430 nm. It was found that illumination with ultraviolet light in the presence of the lysine 13 derivative produced a diminution of the polypeptide of the bc₁ complex having molecular weight 30 000 (band IV) and formation of a new polypeptide composed of band IV and cytochrome c. Band IV was identified as cytochrome c₁, and it was concluded that this hemoprotein interacts with cytochrome c and contains its binding site in complex III of the mitochondrial respiratory chain. Illumination of the bc₁ complex in presence of the lysine 22 derivative did not produce changes of the polypeptide pattern.     

Biogenesis of the cytochrome bc1 complex and role of assembly factors

The cytochrome bc₁ complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc₁ complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc₁ complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc₁ complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc₁ complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc₁ complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Fast oxidation of the primary electron acceptor under anaerobic conditions requires the organization of the photosynthetic chain of Rhodobacter sphaeroides in supercomplexes

The kinetics of reoxidation of the primary acceptor Q_a has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 °C, about half of Q_a⁻ is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q_o of the cytochrome bc₁ complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q_a⁻ oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc₁ complex, which allows a fast transfer of quinone formed at the level of cyt bc₁ complex to the RCs. In agreement with this model, the fast phase of Q_a⁻ reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc₁. The PufX-deleted mutant displays only the slowest phase of Q_a⁻ oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc₁ and RCs.     

Reprint of: Biogenesis of the cytochrome bc1 complex and role of assembly factors

The cytochrome bc₁ complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc₁ complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc₁ complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc₁ complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc₁ complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc₁ complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Ubiquinol oxidation in the cytochrome bc1 complex: Reaction mechanism and prevention of short-circuiting

This review is focused on the mechanism of ubiquinol oxidation by the cytochrome bc₁ complex (bc₁). This integral membrane complex serves as a “hub” in the vast majority of electron transfer chains. The bc₁ oxidizes a ubiquinol molecule to ubiquinone by a unique “bifurcated” reaction where the two released electrons go to different acceptors: one is accepted by the mobile redox active domain of the [2Fe-2S] iron-sulfur Rieske protein (FeS protein) and the other goes to cytochrome b. The nature of intermediates in this reaction remains unclear. It is also debatable how the enzyme prevents short-circuiting that could happen if both electrons escape to the FeS protein. Here, I consider a reaction mechanism that (i) agrees with the available experimental data, (ii) entails three traits preventing the short-circuiting in bc₁, and (iii) exploits the evident structural similarity of the ubiquinone binding sites in the bc₁ and the bacterial photosynthetic reaction center (RC). Based on the latter congruence, it is suggested that the reaction route of ubiquinol oxidation by bc₁ is a reversal of that leading to the ubiquinol formation in the RC. The rate-limiting step of ubiquinol oxidation is then the re-location of a ubiquinol molecule from its stand-by site within cytochrome b into a catalytic site, which is formed only transiently, after docking of the mobile redox domain of the FeS protein to cytochrome b. In the catalytic site, the quinone ring is stabilized by Glu-272 of cytochrome b and His-161 of the FeS protein. The short circuiting is prevented as long as: (i) the formed semiquinone anion remains bound to the reduced FeS domain and impedes its undocking, so that the second electron is forced to go to cytochrome b; (ii) even after ubiquinol is fully oxidized, the reduced FeS domain remains docked to cytochrome b until electron(s) pass through cytochrome b; (iii) if cytochrome b becomes (over)reduced, the binding and oxidation of further ubiquinol molecules is hampered; the reason is that the Glu-272 residue is turned towards the reduced hemes of cytochrome b and is protonated to stabilize the surplus negative charge; in this state, this residue cannot participate in the binding/stabilization of a ubiquinol molecule.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc₁ complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c₁ position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c₁ [S141C(ISP)/G180C(cyt c₁)]. The formation of a disulfide bond between ISP and cyt c₁ in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc₁ complex. Formation of the disulfide bond between ISP and cyt c₁ shortens the distance between the [2Fe-2S] cluster and heme c₁, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.