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.     

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.     

Kinetics of flash-induced electron transfer between bacterial reaction centres,mitochondrial ubiquinol: Cytochrome c oxidoreductase and cytochrome c

Ascorbate-reduced horse heart cytochrome c reduces photo-oxidized bacterial reaction centres with a second-order rate constant of (5–8) · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. In the absence of cytochrome c, the cytochrome c₁ in the ubiquinol:cytochrome c oxidoreductase is oxidized relatively slowly (k = 3.3 · 10⁵ M^?1 · s^?1). Ferrocytochrome c binds specifically to ascorbate-reduced reductase, with a K_d of 0.6 μM, and only the free cytochrome c molecules are involved in the rapid reduction of photo-oxidized reaction centres. The electron transfer between ferricytochrome c and ferrocytochrome c₁ of the reductase is rapid, with a second-order rate constant of 2.1 · 10⁸ M^?1 · s^?1 at an ionic strength of 50 mM. The rate of electron transfer from the Rieske iron-sulphur cluster to cytochrome c₁ is even more rapid. The cytochrome b of the ubiquinol:cytochrome c oxidoreductase can be reduced by electrons from the reaction centres through two pathways: one is sensitive to antimycin and the other to myxothiazol. The amount of cytochrome b reduced in the absence of antimycin is dependent on the redox potential of the system, but in no case tested did it exceed 25% of the amount of photo-oxidized reaction centres.     

Spectral and kinetic resolution of the bc1 complex components in situ: A simple and robust alternative to the traditional difference wavelength approach

The kinetics of the cytochrome (cyt) components of the bc₁ complex (ubiquinol: cytochrome c oxidoreductase, Complex III) are traditionally followed by using the difference of absorbance changes at two or more different wavelengths. However, this difference-wavelength (DW) approach is of limited accuracy in the separation of absorbance changes of components with overlapping spectral bands. To resolve the kinetics of individual components in Rhodobacter sphaeroides chromatophores, we have tested a simplified version of a least squares (LS) analysis, based on measurement at a minimal number of different wavelengths. The success of the simplified LS analysis depended significantly on the wavelengths used in the set. The “traditional” set of 6 wavelengths (542, 551, 561, 566, 569 and 575 nm), normally used in the DW approach to characterize kinetics of cyt c_tot (cyt c₁ + cyt c₂), cyt b_L, cyt b_H, and P870 in chromatophores, could also be used to determine these components via the simplified LS analysis, with improved resolution of the individual components. However, this set is not sufficient when information about cyts c₁ and c₂ is needed. We identified multiple alternative sets of 5 and 6 wavelengths that could be used to determine the kinetics of all 5 components (P870 and cyts c₁, c₂, b_L, and b_H) simultaneously, with an accuracy comparable to that of the LS analysis based on a full set of wavelengths (1 nm intervals). We conclude that a simplified version of LS deconvolution based on a small number of carefully selected wavelengths provides a robust and significant improvement over the traditional DW approach, since it accounts for spectral interference of the different components, and uses fewer measurements when information about all five individual components is needed. Using the simplified and complete LS analyses, we measured the simultaneous kinetics of all cytochrome components of bc₁ complex in the absence and presence of specific inhibitors and found that they correspond well to those expected from the modified Q-cycle. This is the first study in which the kinetics of all cytochrome and reaction center components of the bc₁ complex functioning in situ have been measured simultaneously, with full deconvolution over an extended time range.     

Domain conformational switch of the iron–sulfur protein in cytochrome bc1 complex is induced by the electron transfer from cytochrome bL to bH

Intensive biochemical, biophysical and structural studies of the cytochrome (cyt) bc₁ complex in the past have led to the formulation of the “protonmotive Q-cycle” mechanism for electron and proton transfer in this vitally important complex. The key step of this mechanism is the separation of electrons during the oxidation of a substrate quinol at the Q_P site with both electrons transferred simultaneously to ISP and cyt b_L when the extrinsic domain of ISP (ISP-ED) is located at the b-position. Pre-steady state fast kinetic analysis of bc₁ demonstrates that the reduced ISP-ED moves to the c₁-position to reduce cyt c₁ only after the reduced cyt b_L is oxidized by cyt b_H. However, the question of how the conformational switch of ISP-ED is initiated remains unanswered. The results obtained from analysis of inhibitory efficacy and binding affinity of two types of Q_P site inhibitors, Pm and Pf, under various redox states of the bc₁ complex, suggest that the electron transfer from heme b_L to b_H is the driving force for the releasing of the reduced ISP-ED from the b-position to c₁-position to reduce cyt c₁.     

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.     

The reaction between cytochrome C1 and cytochrome C

The kinetics of electron transfer between the isolated enzymes of cytochrome c₁ and cytochrome c have been investigated using the stopped-flow technique. The reaction between ferrocytochrome c₁ and ferricytochrome c is fast; the second-order rate constant (k₁) is 3.0 · 10⁷ M^?1 · s^?1 at low ionic strength (I = 223 mM, 10°C). The value of this rate constant decreases to 1.8 · 10⁵ M^?1 · s^?1 upon increasing the ionic strength to 1.13 M. The ionic strength dependence of the electron transfer between cytochrome c₁ and cytochrome c implies the involvement of electrostatic interactions in the reaction between both cytochromes. In addition to a general influence of ionic strength, specific anion effects are found for phosphate, chloride and morpholinosulphonate. These anions appear to inhibit the reaction between cytochrome c₁ and cytochrome c by binding of these anions to the cytochrome c molecule. Such a phenomenon is not observed for cacodylate. At an ionic strength of 1.02 M, the second-order rate constants for the reaction between ferrocytochrome c₁ and ferricytochrome c and the reverse reaction are k₁ = 2.4 · 10⁵ M^?1 · s^?1 and k_?1 = 3.3 · 10⁵ M^?1 · s^?1, respectively (450 mM potassium phosphate, pH 7.0, 1% Tween 20, 10°C). The ‘equilibrium’ constant calculated from the rate constants (0.73) is equal to the constant determined from equilibrium studies. Moreover, it is shown that at this ionic strength, the concentrations of intermediary complexes are very low and that the value of the equilibrium constant is independent of ionic strength. These data can be fitted into the following simple reaction scheme: cytochrome c²⁺₁ + cytochrome c³⁺ai cytochrome c³⁺₁ + cytochrome c²⁺.     

Nuclear magnetic resonance study of the rate of electron transfer between cytochrome c and iron hexacyanides

The rates of electron exchange between ferricytochrome c (C^III)3 and ferrocytochrome c (C^II) were observed as a function of the concentrations of ferrihexacyanide (Fe^III) and ferrohexacyanide (Fe^II) by monitoring the line widths of several proton resonances of the protein. Addition of Fe^II to C^III homogeneously increased the line widths of the two downfield paramagnetically shifted heme methyl proton resonances to a maximal value. This was interpreted as indicating the formation of a stoichiometric complex, C^III·Fe^II, in the over-all reaction:

$\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?1}}\text{k}{}_1\text{C}{}^{\mathrm{III}}\text{·}\text{Fe}{}^{\mathrm{II}}\text{?}\text{k}{}_{\mathrm{?2}}\text{k}{}_2\text{C}{}^{\mathrm{II}}\text{·}\text{Fe}{}^{\mathrm{III}}\text{?}\text{k}{}_{\mathrm{?3}}\text{k}{}_3\text{C}{}^{\mathrm{III}}\text{+}\text{Fe}{}^{\mathrm{II}}$

Values for $\text{k}{}_1\text{k}{}_{\mathrm{?1}}\text{= 0.4 × 10}{}^3\text{m}{}^{\mathrm{?1}}\text{and}\text{k}{}_2\text{= 208}\text{s}{}^{\mathrm{?1}}$, respectively, were calculated from the maximal change in line width observed at pH 7.0 and 25 °C. Changes in the line width of C^III in the presence of Fe^II and either KCl or Fe^III suggest that complexation is principally ionic, that Fe^III and Fe^II compete for a common site. Addition of saturating concentrations of Fe^III to C^III produced only minor changes in the nuclear magnetic resonance spectrum of C^III suggesting that complexation occurs on the protein surface.Addition of Fe^III to C^II in the presence of excess Fe^II (to retain most of the protein as C^II) increased the line width of the methyl protons of ligated methionine 80. A value for k_?2 ≈ 2.08 × 10⁴ s^?1 was calculated from the dependence of linewidth on the concentration of Fe^II at 24 °C. These rates are shown to be consistent with the over-all rates of reduction and oxidation previously determined by stopped flow measurements, indicating that k₂ and k_?2 were rate limiting. From the temperature dependence the enthalpies of activation are 7.9 and 15.2 kcal/mol for k₂ and k_?2, respectively.     

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.     

Steady-state kinetics of electron transfer through cytochrome chain of uncoupled submitochondrial particles. II. Influence of pH on kinetics of electron transfer

pH Dependences of steady-state kinetic parameters of cytochrome chains of submitochondrial particles have been studied. It has been shown that the lifetimes of activated states (τ) of the pairs of cytochromes b → c₁ and a → a₃ have different pH dependences; those for the c₁ → c and c → a cytochrome pairs being similar. The rate constants for the non-activated state of the respiratory chains decreased for the b → c₁ pair and increased for the a → a₃ pair when the pH value was increased.The values of pK calculated from these dependences for the pairs b → c₁ and a → a₃ were 7.2 and 8.9, respectively. It has been supposed that the ratio of activated to non-activated electron carriers may be controlled by the local pH value in the mitochondrial membrane, the latter being dependent upon the rate of electron transfer. The kinetic model based on this assumption allows one to explain the experimental dependences on pH of the rate constants for cytochromes b → c, and a → a₃.The values of the diffusion rate constants for H⁺ and OH^? ions in the mitochondrial membrane estimated from these kinetic data obtained in this study weree 10⁴–10⁵ s^?1 and 10²–10³ s^?1, respectively.     

Binding of Imidazole to the Heme of Cytochrome c1 and Inhibition of the bc1 Complex from Rhodobacter sphaeroides: I. EQUILIBRIUM AND MODELING STUDIES*

We have used imidazole (Im) and N-methylimidazole (MeIm) as probes of the heme-binding cavity of membrane-bound cytochrome (cyt) c₁ in detergent-solubilized bc₁ complex from Rhodobacter sphaeroides. Imidazole binding to cyt c₁ substantially lowers the midpoint potential of the heme and fully inhibits bc₁ complex activity. Temperature dependences showed that binding of Im (K_d ≈ 330 μm, 25 °C, pH 8) is enthalpically driven (ΔH⁰ = −56 kJ/mol, ΔS⁰ = −121 J/mol/K), whereas binding of MeIm is 30 times weaker (K_d ≈ 9.3 mm) and is entropically driven (ΔH⁰ = 47 kJ/mol, ΔS⁰° = 197 J/mol/K). The large enthalpic and entropic contributions suggest significant structural and solvation changes in cyt c₁ triggered by ligand binding. Comparison of these results with those obtained previously for soluble cyts c and c₂ suggested that Im binding to cyt c₁ is assisted by formation of hydrogen bonds within the heme cleft. This was strongly supported by molecular dynamics simulations of Im adducts of cyts c, c₂, and c₁, which showed hydrogen bonds formed between the N_δH of Im and the cyt c₁ protein, or with a water molecule sequestered with the ligand in the heme cleft.     

The role of the membrane bound cytochromes of b- and c-type in the electron transport chain of Rhodobacter capsulatus

(1) The electron transport system of heterotrophically dark-grown Rhodobacter capsulatus was investigated using the wild-type strain MT1131 and the phototrophic non-competent (Ps^-) mutant MT-GS18 carrying deletions of the genes for cytochrome c ₁ and b of the bc ₁ complex and for cytochrome c ₂. (2) Spectroscopic and thermodynamic data demonstrate that deletion of both bc ₁ complex and cyt. c ₂ still leaves several haems of c- and b-type with Em_7.0 of +265 mV and +354 mV at 551–542 nm, and +415 mV and +275 mV at 561–575 nm, respectively. (3) Analysis of the oxidoreduction kinetic patterns of cytochromes indicated that cyt. b ₄₁₅ and cyt. b ₂₇₅ are reduced by either ascorbate-diaminodurene or NADH, respectively. (4) Growth on different carbon and nitrogen sources revealed that the membrane-bound electron transport chain of both MT1131 and MT-GS18 strains undergoes functional modifications in response to the composition of the growth medium used. (5) Excitation of membrane fragments from cells grown in malate minimal medium by a train of single turnover flashes of light led to a rapid oxidation of 32% of the membrane-bound c-type haem complement. Conversely, membranes prepared from peptone/yeast extract grown cells did not show cyt. c photooxidation. These results are discussed within the framework of an electron transport chain in which alternative pathways bypassing both the cyt. c ₂ and bc ₁ complex might involve high-potential membrane bound haems of b- and c-type.Abbreviations AA antimycin A - CCCP carbonylcyanide m-chlorophenyl hydrazone - CN^- cyanide - DAD diaminodurene - Q₂H₂ ubiquinol-2 - Q-pool ubiquinone-10 pool - RC photochemical reaction center     

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.     

Gas hold-up and oxygen mass transfer in three pneumatic bioreactors operating with sugarcane bagasse suspensions

Sugarcane bagasse is a low-cost and abundant by-product generated by the bioethanol industry, and is a potential substrate for cellulolytic enzyme production. The aim of this work was to evaluate the effects of air flow rate (Q _AIR), solids loading (%_S), sugarcane bagasse type, and particle size on the gas hold-up (ε _G) and volumetric oxygen transfer coefficient (k _L a) in three different pneumatic bioreactors, using response surface methodology. Concentric tube airlift (CTA), split-cylinder airlift (SCA), and bubble column (BC) bioreactor types were tested. Q _AIR and  %_S affected oxygen mass transfer positively and negatively, respectively, while sugarcane bagasse type and particle size (within the range studied) did not influence k _L a. Using large particles of untreated sugarcane bagasse, the loop-type bioreactors (CTA and SCA) exhibited higher mass transfer, compared to the BC reactor. At higher  %_S, SCA presented a higher k _L a value (0.0448 s^?1) than CTA, and the best operational conditions in terms of oxygen mass transfer were achieved for  %_S < 10.0 g L^?1 and Q _AIR > 27.0 L min^?1. These results demonstrated that pneumatic bioreactors can provide elevated oxygen transfer in the presence of vegetal biomass, making them an excellent option for use in three-phase systems for cellulolytic enzyme production by filamentous fungi.     

Influences of substrates of different microsomal electron transfer pathways on the oxidation-reduction kinetics of microsomal cytochrome b5.

(i) Compounds activating the microsomal electron transfer oxidative reactions, e.g., the mixed function oxidase (aminopyrine, aniline), the Δ⁹-desaturase (stearyl-CoA), and lipid peroxidation reaction (iron pyrophosphate), cause a decrease in the steady-state reduced level of cytochrome b₅. (ii) In the absence of substrates, the k_ox for cytochrome b₅ was the same whether reduced by NADH or NADPH (about 0.045 S^?1, indicating that no distinction exists between the cytochrome b₅ involved in NADH-driven and NADPH-driven microsomal reactions which utilize this hemoprotein. (iii) The agents activating the oxidative pathways affect the first-order rate constant for cytochrome b₅ oxidation (k_ox), but the apparent first-order rate constant obtained for reduction (k_red) of cytochrome b₅ by NADPH is still more than 10 times the k_ox, and the k_red obtained with NADH is still more than 100 times the k_ox. (iv) Of the compounds used, only stearyl-CoA caused a decrease in the NADH-supported steady-state reduced level of cytochrome b₅. This effect is probably due to a detergent-like action of stearyl-CoA on the membrane proteins, interfering with some interactions (e.g., NADPH-cytochrome c reductase with cytochrome P-450; NADH-cytochrome b₅ reductase with cytochrome b₅). (v) Based upon the kinetic and steady-state measurements it is concluded that substrate-induced changes in the steady-state reduced level of cytochrome b₅ are evidence for a decrease in the population of this hemoprotein available to the reductase due to competition with other more favored acceptors, (vi) Measurements using the duration of the reduced state and rates of electron flow through cytochrome b₅ reveal that normally about 60% of the NADH-derived reducing equivalents go through cytochrome b₅ while only about one electron in nine passes through this cytochrome when NADPH is the source of reducing equivalents. Substrates of the various pathways alter the proportion of electrons passing through cytochrome b₅ depending upon their activating or inhibiting action on cytochrome b₅-dependent or -independent reactions.     

Studies on Inhibition of Respiratory Cytochrome bc 1 Complex by the Fungicide Pyrimorph Suggest a Novel Inhibitory Mechanism

The respiratory chain cytochrome bc ₁ complex (cyt bc ₁) is a major target of numerous antibiotics and fungicides. All cyt bc ₁ inhibitors act on either the ubiquinol oxidation (Q_P) or ubiquinone reduction (Q_N) site. The primary cause of resistance to bc ₁ inhibitors is target site mutations, creating a need for novel agents that act on alternative sites within the cyt bc ₁ to overcome resistance. Pyrimorph, a synthetic fungicide, inhibits the growth of a broad range of plant pathogenic fungi, though little is known concerning its mechanism of action. In this study, using isolated mitochondria from pathogenic fungus Phytophthora capsici, we show that pyrimorph blocks mitochondrial electron transport by affecting the function of cyt bc ₁. Indeed, pyrimorph inhibits the activities of both purified 11-subunit mitochondrial and 4-subunit bacterial bc ₁ with IC₅₀ values of 85.0 μM and 69.2 μM, respectively, indicating that it targets the essential subunits of cyt bc ₁ complexes. Using an array of biochemical and spectral methods, we show that pyrimorph acts on an area near the Q_P site and falls into the category of a mixed-type, noncompetitive inhibitor with respect to the substrate ubiquinol. In silico molecular docking of pyrimorph to cyt b from mammalian and bacterial sources also suggests that pyrimorph binds in the vicinity of the quinol oxidation site.     

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 Q_o 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 Q_o 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 Q_o 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.     

Cloning,heterologous expression,and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris

The cytochrome (cyt) c′, cyt c₅₅₆, and cyt c₂ genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c′ and cyt c₅₅₆ have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b₅₆₂, in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe^II and Fe^III states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe^II-cyt c₅₅₆ is more stable than the corresponding state of Fe^III-cyt c₅₅₆ (ΔΔG_f°=22 kJ/mol).     

Reactions of mercaptans with cytochrome c oxidase and cytochrome c

1. The steady-state oxidation of ferrocytochrome c by dioxygen catalyzed by cytochrome c oxidase, is inhibited non-competitively towards cytochrome c by methanethiol, ethanethiol, 1-propanethiol and 1-butanethiol with K_i values of 4.5, 91, 200 and 330 μM, respectively.2. The inhibition constant K_i of ethanethiol is found to be constant between pH 5 and 8, which suggests that only the neutral form of the thiol inhibits the enzyme.3. The absorption spectrum of oxidized cytochrome c oxidase in the Soret region shows rapid absorbance changes upon addition of ethanethiol to the enzyme. This process is followed by a very slow reduction of the enzyme. The fast reaction, which represents a binding reaction of ethanethiol to cytochrome c oxidase, has a k₁ of 33 M^?1 · s^?1 and dissociation constant K_d of 3.9 mM.4. Ethanethiol induces fast spectral changes in the absorption spectrum of cytochrome c, which are followed by a very slow reduction of the heme. The rate constant for the fast ethanethiol reaction representing a bimolecular binding step is 50 M^?1 · s^?1 and the dissociation constant is about 2 mM. Addition of up to 25 mM ethanethiol to ferrocytochrome c does not cause spectral changes.5. EPR (electron paramagnetic resonance) spectra of cytochrome c oxidase, incubated with methanethiol or ethanethiol in the presence of cytochrome c and ascorbate, show the formation of low-spin cytochrome a₃-mercaptide compounds with g values of 2.39, 2.23, 1.93 and of 2.43, 2.24, 1.91, respectively.     

Role of phospholipids in respiratory cytochrome bc1 complex catalysis and supercomplex formation

Specific protein-lipid interactions have been identified in X-ray structures of membrane proteins. The role of specifically bound lipid molecules in protein function remains elusive. In the current study, we investigated how phospholipids influence catalytic, spectral and electrochemical properties of the yeast respiratory cytochrome bc₁ complex and how disruption of a specific cardiolipin binding site in cytochrome c₁ alters respiratory supercomplex formation in mitochondrial membranes. Purified yeast cytochrome bc₁ complex was treated with phospholipase A₂. The lipid-depleted enzyme was stable but nearly catalytically inactive. The absorption maxima of the reduced b-hemes were blue-shifted. The midpoint potentials of the b-hemes of the delipidated complex were shifted from − 52 to − 82 mV (heme b_L) and from + 113 to − 2 mV (heme b_H). These alterations could be reversed by reconstitution of the delipidated enzyme with a mixture of asolectin and cardiolipin, whereas addition of the single components could not reverse the alterations. We further analyzed the role of a specific cardiolipin binding site (CL_i) in supercomplex formation by site-directed mutagenesis and BN-PAGE. The results suggested that cardiolipin stabilizes respiratory supercomplex formation by neutralizing the charges of lysine residues in the vicinity of the presumed interaction domain between cytochrome bc₁ complex and cytochrome c oxidase. Overall, the study supports the idea, that enzyme-bound phospholipids can play an important role in the regulation of protein function and protein-protein interaction.