Identification of hydrogen bonding network for proton transfer at the quinol oxidation site of Rhodobacter capsulatus cytochrome bc1.

Cytochrome bc1 catalyzes electron transfer from quinol (QH2) to cytochrome c in reactions coupled to proton translocation across the energy-conserving membrane. Energetic efficiency of the catalytic cycle is secured by a two-electron and two-proton bifurcation reaction leading to oxidation of QH2 and reduction of the Rieske cluster and heme bL. The proton paths associated with this reaction remain elusive. Here, we used site-directed mutagenesis and quantum mechanical calculations to analyze the contribution of protonable side chains located at the heme bL side of the QH2 oxidation site in Rhodobacter capsulatus cytochrome bc1. We observe that the proton path is effectively switched off when H276 and E295 are simultaneously mutated to the nonprotonable residues in the H276F/E295V double mutant. The two single mutants, H276F or E295V, are less efficient but still transfer protons at functionally relevant rates. Natural selection exposed two single mutations, N279S and M154T, that restored the functional proton transfers in H276F/E295V. Quantum mechanical calculations indicated that H276F/E295V traps the side chain of Y147 in a position distant from QH2, whereas either N279S or M154T induce local changes releasing Y147 from that position. This shortens the distance between the protonable groups of Y147 and D278 and/or increases mobility of the Y147 side chain, which makes Y147 efficient in transferring protons from QH2 toward D278 in H276F/E295V. Overall, our study identified an extended hydrogen bonding network, build up by E295, H276, D278, and Y147, involved in efficient proton removal from QH2 at the heme bL side of QH2 oxidation site.

On the inter-monomer electron transfer in cytochrome bc1.

Cytochrome bc1 is a structural and functional homodimer. The catalytically-relevant inter-monomer electron transfer has been implicated by a number of experiments, including those based on analyses of the cross-dimer mutated derivatives. As some of the original data on these derivatives have recently been questioned, we extend kinetic analysis of these mutants to confirm the enzymatic origin of the observed activities and their relevance in exploration of conditions that expose electron transfer between the monomers. While obtained data consistently implicate rapid inter-monomer electron equilibration in cytochrome bc1, the mechanistic and physiological meaning of this equilibration is yet to be established.

Redox Active Antimicrobial Peptides in Controlling Growth of Microorganisms at Body Barriers.

Epithelia in the skin, gut and other environmentally exposed organs display a variety of mechanisms to control microbial communities and limit potential pathogenic microbial invasion. Naturally occurring antimicrobial proteins/peptides and their synthetic derivatives (here collectively referred to as AMPs) reinforce the antimicrobial barrier function of epithelial cells. Understanding how these AMPs are functionally regulated may be important for new therapeutic approaches to combat microbial infections. Some AMPs are subject to redox-dependent regulation. This review aims to: (i) explore cysteine-based redox active AMPs in skin and intestine; (ii) discuss casual links between various redox environments of these barrier tissues and the ability of AMPs to control cutaneous and intestinal microbes; (iii) highlight how bacteria, through intrinsic mechanisms, can influence the bactericidal potential of redox-sensitive AMPs.

Catalytic Reactions and Energy Conservation in the Cytochrome bc1 and b6f Complexes of Energy-Transducing Membranes.

This review focuses on key components of respiratory and photosynthetic energy-transduction systems: the cytochrome bc1 and b6f (Cytbc1/b6f) membranous multisubunit homodimeric complexes. These remarkable molecular machines catalyze electron transfer from membranous quinones to water-soluble electron carriers (such as cytochromes c or plastocyanin), coupling electron flow to proton translocation across the energy-transducing membrane and contributing to the generation of a transmembrane electrochemical potential gradient, which powers cellular metabolism in the majority of living organisms. Cytsbc1/b6f share many similarities but also have significant differences. While decades of research have provided extensive knowledge on these enzymes, several important aspects of their molecular mechanisms remain to be elucidated. We summarize a broad range of structural, mechanistic, and physiological aspects required for function of Cytbc1/b6f, combining textbook fundamentals with new intriguing concepts that have emerged from more recent studies. The discussion covers but is not limited to (i) mechanisms of energy-conserving bifurcation of electron pathway and energy-wasting superoxide generation at the quinol oxidation site, (ii) the mechanism by which semiquinone is stabilized at the quinone reduction site, (iii) interactions with substrates and specific inhibitors, (iv) intermonomer electron transfer and the role of a dimeric complex, and (v) higher levels of organization and regulation that involve Cytsbc1/b6f. In addressing these topics, we point out existing uncertainties and controversies, which, as suggested, will drive further research in this field.

ROS signaling capacity of cytochrome bc1: Opposing effects of adaptive and pathogenic mitochondrial mutations.

Cytochrome bc1, also known as mitochondrial complex III, is considered to be one of the important producers of reactive oxygen species (ROS) in living organisms. Under physiological conditions, a certain level of ROS produced by mitochondrial electron transport chain (ETC) might be beneficial and take part in cellular signaling. However, elevated levels of ROS might exhibit negative effects, resulting in cellular damage. It is well known that inhibiting the electron flow within mitochondrial complex III leads to high production of ROS. However, superoxide production by cytochrome bc1 in a non-inhibited system remained controversial. Here, we propose a novel method for ROS detection in ETC hybrid system in solution comprising bacterial cytochrome bc1 and mitochondrial complex IV. We clearly show that non-inhibited cytochrome bc1 generates ROS and that adaptive and pathogenic mitochondrial mutations suppress and enhance ROS production, respectively. We also noted that cytochrome bc1 produces ROS in a rate-dependent manner and that the mechanism of ROS generation changes according to the rate of operation of the enzyme. This dependency has not yet been reported, but seems to be crucial when discussing ROS signaling originating from mitochondria.

The antimicrobial activity of chemerin-derived peptide p4 requires oxidative conditions.

Chemerin is a leukocyte attractant, adipokine, and antimicrobial protein abundantly produced in the skin epidermis. Despite the fact that most of the bactericidal activity present in human skin exudates is chemerin-dependent, just how chemerin shapes skin defenses remains obscure. Here we demonstrate that p4, a potent antimicrobial human chemerin peptide derivative, displays killing activity against pathogenic methicillin-resistant Staphylococcus aureus strains and suppresses microbial growth in a topical skin infection model. Mechanistically, we show that p4 homodimerization is required for maximal bactericidal activity and that an oxidative environment, such as at the skin surface, facilitates p4 disulfide bridge formation, required for the dimerization. p4 led to rapid damage of the bacterial internal membrane and inhibited the interaction between the membranous cytochrome bc1 complex and its redox partner, cytochrome c These results suggest that a chemerin p4-based defense strategy combats bacterial challenges at the skin surface.

Suppression of superoxide production by a spin-spin coupling between semiquinone and the Rieske cluster in cytochrome bc1.

Catalytic reactions of quinol oxidoreductases may lead to the generation of superoxide due to electron leaks from unstable semiquinone intermediates (SQ). For cytochrome bc1 , the mechanism of suppression of superoxide generation remains unknown. We analyzed conditions of formation of a spin-spin-coupled state between SQ and the Rieske cluster (SQ-FeS) associated with catalysis of the quinol oxidation site of cytochrome bc1 . We reveal that mutants that preclude direct interaction between SQ and the Rieske cluster do not form SQ-FeS and release enhanced superoxide. In the enzymes generating SQ-FeS, little or no superoxide is detected. We propose that SQ-FeS suppresses superoxide generation, becoming an element modulating superoxide release under physiologically relevant conditions slowing electron flow through the enzyme.

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b, iron-sulfur protein and cytochrome c1.

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc1. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Qo site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c1, iii) reduction of cytochrome c1 by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c1. In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c1 and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c1 of cytochrome c1, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc1 were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Qo site and cytochrome c1 helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Qo site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.

Mitochondrial disease-related mutations at the cytochrome b-iron-sulfur protein (ISP) interface: Molecular effects on the large-scale motion of ISP and superoxide generation studied in Rhodobacter capsulatus cytochrome bc1.

One of the important elements of operation of cytochrome bc1 (mitochondrial respiratory complex III) is a large scale movement of the head domain of iron-sulfur protein (ISP-HD), which connects the quinol oxidation site (Qo) located within the cytochrome b, with the outermost heme c(1) of cytochrome c(1). Several mitochondrial disease-related mutations in cytochrome b are located at the cytochrome b-ISP-HD interface, thus their molecular effects can be associated with altered motion of ISP-HD. Using purple bacterial model, we recently showed that one of such mutations - G167P shifts the equilibrium position of ISP-HD towards positions remote from the Qo site as compared to the native enzyme [Borek et al., J. Biol. Chem. 290 (2015) 23781-23792]. This resulted in the enhanced propensity of the mutant to generate reactive oxygen species (ROS) which was explained on the basis of the model evoking "semireverse" electron transfer from heme bL to quinone. Here we examine another mutation from that group - G332D (G290D in human), finding that it also shifts the equilibrium position of ISP-HD in the same direction, however displays less of the enhancement in ROS production. We provide spectroscopic indication that G332D might affect the electrostatics of interaction between cytochrome b and ISP-HD. This effect, in light of the measured enzymatic activities and electron transfer rates, appears to be less severe than structural distortion caused by proline in G167P mutant. Comparative analysis of the effects of G332D and G167P confirms a general prediction that mutations located at the cytochrome b-ISP-HD interface influence the motion of ISP-HD and indicates that "pushing" ISP-HD away from the Qo site is the most likely outcome of this influence. It can also be predicted that an increase in ROS production associated with the "pushing" effect is quite sensitive to overall severity of this change with more active mutants being generally more protected against elevated ROS. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

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 hemesb The hemes display a large difference in redox midpoint potentials (ΔEm_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 hemebligand mutants of cytochromebc1in 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 ΔEm_b can be markedly increased, and the redox potential of one of the hemes can stay above the level of quinone pool, or ΔEm_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 functionalin vivo This reveals that cytochromebc1can accommodate large changes in ΔEm_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 hemesbin this cytochrome and in other membranous cytochromesbact as electronic connectors for the catalytic sites with no fine tuning in ΔEm_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.

[Molecular effects of mitochondrial mutations in cytochrome b of complex III and their impact on the levels of free radical production]. / Molekularne efekty mutacji mitochondrialnych w genie kodujacym cytochrom b kompleksu III i ich wplyw na poziom produkcji wolnych rodników.

Cytochrome bc1 (mitochondrial complex III) is a common element of several bioenergetic systems. This enzyme catalyses electron transfer from ubiquinol to cytochrome c coupled to translocation of protons across the membrane, which contributes to generation of protonmotive force utilized for ATP production. Cytochrome b, together with cytochrome c1 and iron-sulfur protein (ISP), forms the evolutionarily conserved catalytic core. Transfer of electrons within this enzyme, is facilitated by the movement of ISP domain that allows communication between cytochrome b and cytochrome c1. Mutations in the subunits of catalytic core may cause mitochondrial diseases, however elucidation of their molecular effects in human cells is difficult. For that reason yeast or bacterial systems are used. It was found that some mutations in cytochrome b influence the movement of ISP and, in consequence, the levels of superoxide generation. By exploring the effects of mitochondrial mutations in model systems one can not only learn about molecular basis of diseases but also gain insights about catalytic and side reactions in cytochrome bc1.

Effect of H bond removal and changes in the position of the iron-sulphur head domain on the spin-lattice relaxation properties of the [2Fe-2S](2+) Rieske cluster in cytochrome bc(1).

Here, comparative electron spin-lattice relaxation studies of the 2Fe-2S iron-sulphur (Fe-S) cluster embedded in a large membrane protein complex - cytochrome bc1 - are reported. Structural modifications of the local environment alone (mutations S158A and Y160W removing specific H bonds between Fe-S and amino acid side chains) or in combination with changes in global protein conformation (mutations/inhibitors changing the position of the Fe-S binding domain within the protein complex) resulted in different redox potentials as well as g-, g-strain and the relaxation rates (T1(-1)) for the Fe-S cluster. The relaxation rates for T < 25 K were measured directly by inversion recovery, while for T > 60 K they were deduced from simulation of continuous wave EPR spectra of the cluster using a model that included anisotropy of Lorentzian broadening. In all cases, the relaxation rate involved contributions from direct, second-order Raman and Orbach processes, each dominating over different temperature ranges. The analysis of T1(-1) (T) over the range 5-120 K yielded the values of the Orbach energy (EOrb), Debye temperature θD and Raman process efficiency CRam for each variant of the protein. As the Orbach energy was generally higher for mutants S158A and Y160W, compared to wild-type protein (WT), it is suggested that H bond removal influences the geometry leading to increased strength of antiferromagnetic coupling between two Fe ions of the cluster. While θD was similar for all variants (∼107 K), the efficiency of the Raman process generally depends on the spin-orbit coupling that is lower for S158A and Y160W mutants, when compared to the WT. However, in several cases CRam did not only correlate with spin-orbit coupling but was also influenced by other factors - possibly the modification of protein rigidity and therefore the vibrational modes around the Fe-S cluster that change upon the movement of the iron-sulphur head domain.

Mitochondrial Disease-related Mutation G167P in Cytochrome b of Rhodobacter capsulatus Cytochrome bc1 (S151P in Human) Affects the Equilibrium Distribution of [2Fe-2S] Cluster and Generation of Superoxide.

Cytochrome bc1 is one of the key enzymes of many bioenergetic systems. Its operation involves a large scale movement of a head domain of iron-sulfur protein (ISP-HD), which functionally connects the catalytic quinol oxidation Qo site in cytochrome b with cytochrome c1. The Qo site under certain conditions can generate reactive oxygen species in the reaction scheme depending on the actual position of ISP-HD in respect to the Qo site. Here, using a bacterial system, we show that mutation G167P in cytochrome b shifts the equilibrium distribution of ISP-HD toward positions remote from the Qo site. This renders cytochrome bc1 non-functional in vivo. This effect is remediated by addition of alanine insertions (1Ala and 2Ala) in the neck region of the ISP subunit. These insertions, which on their own shift the equilibrium distribution of ISP-HD in the opposite direction (i.e. toward the Qo site), also act in this manner in the presence of G167P. Changes in the equilibrium distribution of ISP-HD in G167P lead to an increased propensity of cytochrome bc1 to generate superoxide, which becomes evident when the concentration of quinone increases. This result corroborates the recently proposed model in which "semireverse" electron transfer back to the Qo site, occurring when ISP-HD is remote from the site, favors reactive oxygen species production. G167P suggests possible molecular effects of S151P (corresponding in sequence to G167P) identified as a mitochondrial disease-related mutation in human cytochrome b. These effects may be valid for other human mutations that change the equilibrium distribution of ISP-HD in a manner similar to G167P.

Catalytically-relevant electron transfer between two hemes bL in the hybrid cytochrome bc1-like complex containing a fusion of Rhodobacter sphaeroides and capsulatus cytochromes b.

To address mechanistic questions about the functioning of dimeric cytochrome bc1 new genetic approaches have recently been developed. They were specifically designed to enable construction of asymmetrically-mutated variants suitable for functional studies. One approach exploited a fusion of two cytochromes b that replaced the separate subunits in the dimer. The fusion protein, built from two copies of the same cytochrome b of purple bacterium Rhodobacter capsulatus, served as a template to create a series of asymmetrically-mutated cytochrome bc1-like complexes (B-B) which, through kinetic studies, disclosed several important principles of dimer engineering. Here, we report on construction of another fusion protein complex that adds a new tool to investigate dimeric function of the enzyme through the asymmetrically mutated forms of the protein. This complex (BS-B) contains a hybrid protein that combines two different cytochromes b: one coming from R. capsulatus and the other - from a closely related species, R. sphaeroides. With this new fusion we addressed a still controversial issue of electron transfer between the two hemes bL in the core of dimer. Kinetic data obtained with a series of BS-B variants provided new evidence confirming the previously reported observations that electron transfer between those two hemes occurs on a millisecond timescale, thus is a catalytically-relevant event. Both types of the fusion complexes (B-B and BS-B) consistently implicate that the heme-bL-bL bridge forms an electronic connection available for inter-monomer electron transfer in cytochrome bc1.

Enzymatic activities of isolated cytochrome bc1-like complexes containing fused cytochrome b subunits with asymmetrically inactivated segments of electron transfer chains.

Homodimeric structure of cytochrome bc1, a common component of biological energy conversion systems, builds in four catalytic quinone oxidation/reduction sites and four chains of cofactors (branches) that, connected by a centrally located bridge, form a symmetric H-shaped electron transfer system. The mechanism of operation of this complex system is under constant debate. Here, we report on isolation and enzymatic examination of cytochrome bc1-like complexes containing fused cytochrome b subunits in which asymmetrically introduced mutations inactivated individual branches in various combinations. The structural asymmetry of those forms was confirmed spectroscopically. All the asymmetric forms corresponding to cytochrome bc1 with partial or full inactivation of one monomer retain high enzymatic activity but at the same time show a decrease in the maximum turnover rate by a factor close to 2. This strongly supports the model assuming independent operation of monomers. The cross-inactivated form corresponding to cytochrome bc1 with disabled complementary parts of each monomer retains the enzymatic activity at the level that, for the first time on isolated from membranes and purified to homogeneity preparations, demonstrates that intermonomer electron transfer through the bridge effectively sustains the enzymatic turnover. The results fully support the concept that electrons freely distribute between the four catalytic sites of a dimer and that any path connecting the catalytic sites on the opposite sides of the membrane is enzymatically competent. The possibility to examine enzymatic properties of isolated forms of asymmetric complexes constructed using the cytochrome b fusion system extends the array of tools available for investigating the engineering of dimeric cytochrome bc1 from the mechanistic and physiological perspectives.

Fusing two cytochromes b of Rhodobacter capsulatus cytochrome bc1 using various linkers defines a set of protein templates for asymmetric mutagenesis.

Cytochrome bc(1) (mitochondrial complex III), one of the key enzymes of biological energy conversion, is a functional homodimer in which each monomer contains three catalytic subunits: cytochrome c(1), the iron-sulfur subunit and cytochrome b. The latter is composed of eight transmembrane α-helices which, in duplicate, form a hydrophobic core of a dimer. We show that two cytochromes b can be fused into one 16-helical subunit using a number of different peptide linkers that vary in length but all connect the C-terminus of one cytochrome with the N-terminus of the other. The fusion proteins replace two cytochromes b in the dimer defining a set of available protein templates for introducing mutations that allow breaking symmetry of a dimer. A more detailed comparison of the form with the shortest, 3 amino acid, linker to the form with 12 amino acid linker established that both forms display similar level of structural plasticity to accommodate several, but not all, asymmetric patterns of mutations that knock out individual segments of cofactor chains. While the system based on a fused gene does not allow for the assessments of the functionality of electron-transfer paths in vivo, the family of proteins with fused cytochrome b offers attractive model for detailed investigations of molecular mechanism of catalysis at in vitro/reconstitution level.

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1. Implications for the mechanism of superoxide production.

In addition to its bioenergetic function of building up proton motive force, cytochrome bc1 can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ(o)) formed at the quinone oxidation/reduction Q(o) site (Q(o)) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b(L) (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc1 that severely impeded the oxidation of FeS by cytochrome c1, changed density of FeS around Q(o) by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q(o) and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ(o) that present a risk of generation of superoxide by cytochrome bc1. Isolation of this reaction sequence from multiplicity of possible reactions at Q(o) helps to better understand conditions under which complex III might contribute to ROS generation in vivo.

An electronic bus bar lies in the core of cytochrome bc1.

The ubiquinol-cytochrome c oxidoreductases, central to cellular respiration and photosynthesis, are homodimers. High symmetry has frustrated resolution of whether cross-dimer interactions are functionally important. This has resulted in a proliferation of contradictory models. Here, we duplicated and fused cytochrome b subunits, and then broke symmetry by introducing independent mutations into each monomer. Electrons moved freely within and between monomers, crossing an electron-transfer bridge between two hemes in the core of the dimer. This revealed an H-shaped electron-transfer system that distributes electrons between four quinone oxidation-reduction terminals at the corners of the dimer within the millisecond time scale of enzymatic turnover. Free and unregulated distribution of electrons acts like a molecular-scale bus bar, a design often exploited in electronics.

Movement of the iron-sulfur head domain of cytochrome bc(1) transiently opens the catalytic Q(o) site for reaction with oxygen.

Cytochrome bc(1), a key enzyme of biological energy conversion, generates or uses a proton motive force through the Q cycle that operates within the two chains of cofactors that embed two catalytic quinone oxidation/reduction sites, the Q(o) site and the Q(i) site. The Q(o) site relies on the joint action of two cofactors, the iron-sulfur (FeS) cluster and heme b(L). Side reactions of the Q cycle involve a generation of superoxide which is commonly thought to be a product of an oxidation of a highly unstable semiquinone formed in the Q(o) site (SQ(o)), but the overall mechanism of superoxide generation remains poorly understood. Here, we use selectively modified chains of cytochrome bc(1) to clearly isolate states linked with superoxide production. We show that this reaction takes place under severely impeded electron flow that traps heme b(L) in the reduced state and reflects a probability with which a single electron on SQ(o) is capable of reducing oxygen. SQ(o) gains this capability only when the FeS head domain, as a part of a catalytic cycle, transiently leaves the Q(o) site to communicate with the outermost cofactor, cytochrome c(1). This increases the distance between the FeS cluster and the remaining portion of the Q(o) site, reducing the likelihood that the FeS cluster participates in an immediate removal of SQ(o). In other states, the presence of both the FeS cluster and heme b(L) in the Q(o) site increases the probability of completion of short-circuit reactions which retain single electrons within the enzyme instead of releasing them on oxygen. We propose that in this way, cytochrome bc(1) under conditions of impeded electron flow employs the leak-proof short-circuits to minimize the unwanted single-electron reduction of oxygen.

Demonstration of short-lived complexes of cytochrome c with cytochrome bc1 by EPR spectroscopy: implications for the mechanism of interprotein electron transfer.

One of the steps of a common pathway for biological energy conversion involves electron transfer between cytochrome c and cytochrome bc1. To clarify the mechanism of this reaction, we examined the structural association of those two proteins using the electron transfer-independent electron paramagnetic resonance (EPR) techniques. Drawing on the differences in the continuous wave EPR spectra and saturation recoveries of spin-labeled bacterial and mitochondrial cytochromes c recorded in the absence and presence of bacterial cytochrome bc1, we have exposed a time scale of dynamic equilibrium between the bound and the free state of cytochrome c at various ionic strengths. Our data show a successive decrease of the bound cytochrome c fraction as the ionic strength increases, with a limit of approximately 120 mm NaCl above which essentially no bound cytochrome c can be detected by EPR. This limit does not apply to all of the interactions of cytochrome c with cytochrome bc1 because the cytochrome bc1 enzymatic activity remained high over a much wider range of ionic strengths. We concluded that EPR monitors just the tightly bound state of the association and that an averaged lifetime of this state decreases from over 100 micros at low ionic strength to less than 400 ns at an ionic strength above 120 mm. This suggests that at physiological ionic strength, the tightly bound complex on average lasts less than the time needed for a single electron exchange between hemes c and c1, indicating that productive electron transfer requires several collisions of the two molecules. This is consistent with an early idea of diffusion-coupled reactions that link the soluble electron carriers with the membranous complexes, which, we believe, provides a robust means of regulating electron flow through these complexes.