The cytochrome b Zn binding amino acid residue histidine 291 is essential for ubihydroquinone oxidation at the Qo site of bacterial cytochrome bc1.

The ubiquinol:cytochrome (cyt) c oxidoreductase (or cyt bc1) is an important membrane protein complex in photosynthetic and respiratory energy transduction. In bacteria such as Rhodobacter capsulatus it is constituted of three subunits: the iron-sulfur protein, cyt b and cyt c1, which form two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo site, the pathways of bifurcated electron transfers emanating from QH2 oxidation are known, but the associated proton release routes are not well defined. In energy transducing complexes, Zn2+ binding amino acid residues often correlate with proton uptake or release pathways. Earlier, using combined EXAFS and structural studies, we identified Zn coordinating residues of mitochondrial and bacterial cyt bc1. In this work, using the genetically tractable bacterial cyt bc1, we substituted each of the proposed Zn binding residues with non-protonatable side chains. Among these mutants, only the His291Leu substitution destroyed almost completely the Qo site catalysis without perturbing significantly the redox properties of the cofactors or the assembly of the complex. In this mutant, which is unable to support photosynthetic growth, the bifurcated electron transfer reactions that result from QH2 oxidation at the Qo site, as well as the associated proton(s) release, were dramatically impaired. Based on these findings, on the putative role of His291 in liganding Zn, and on its solvent exposed and highly conserved position, we propose that His291 of cyt b is critical for proton release associated to QH2 oxidation at the Qo site of cyt bc1.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Cardiolipin-based respiratory complex activation in bacteria.

Anionic lipids play a variety of key roles in membrane function, including functional and structural effects on respiratory complexes. However, little is known about the molecular basis of these lipid-protein interactions. In this study, NarGHI, an anaerobic respiratory complex of Escherichia coli, has been used to investigate the relations in between membrane-bound proteins with phospholipids. Activity of the NarGHI complex is enhanced by anionic phospholipids both in vivo and in vitro. The anionic cardiolipin tightly associates with the NarGHI complex and is the most effective phospholipid to restore functionality of a nearly inactive detergent-solubilized enzyme complex. A specific cardiolipin-binding site is identified on the basis of the available X-ray diffraction data and of site-directed mutagenesis experiment. One acyl chain of cardiolipin is in close proximity to the heme b(D) center and is responsible for structural adjustments of b(D) and of the adjacent quinol substrate binding site. Finally, cardiolipin binding tunes the interaction with the quinol substrate. Together, our results provide a molecular basis for the activation of a bacterial respiratory complex by cardiolipin.

A robust genetic system for producing heterodimeric native and mutant cytochrome bc(1).

The ubihydroquinone:cytochrome c oxidoreductase, or cytochrome bc1, is central to the production of ATP by oxidative phosphorylation and photophosphorylation in many organisms. Its three-dimensional structure depicts it as a homodimer with each monomer composed of the Fe-S protein, cytochrome b, and cytochrome c1 subunits. Recent genetic approaches successfully produced heterodimeric variants of this enzyme, providing insights into its mechanism of function. However, these experimental setups are inherently prone to genetic rearrangements as they carry repeated copies of cytochrome bc1 structural genes. Duplications present on a single replicon (one-plasmid system) or a double replicon (two-plasmid system) could yield heterogeneous populations via homologous recombination or other genetic events at different frequencies, especially under selective growth conditions. In this work, we assessed the origins and frequencies of genetic variations encountered in these systems and describe an improved variant of the two-plasmid system. We found that use of a recombination-deficient background (recA) minimizes spontaneous formation of co-integrant plasmids and renders the homologous recombination within the cytochrome b gene copies inconsequential. On the basis of the data, we conclude that both the newly improved RecA-deficient and the previously used RecA-proficient two-plasmid systems reliably produce native and mutant heterodimeric cytochrome bc1 variants. The two-plasmid system developed here might contribute to the study of "mitochondrial heteroplasmy"-like heterogeneous states in model bacteria (e.g., Rhodobacter species) suitable for bioenergetics studies. In the following paper (DOI 10.1021/bi400561e), we describe the use of the two-plasmid system to produce and characterize, in membranes and in purified states, an active heterodimeric cytochrome bc1 variant with unusual intermonomer electron transfer properties.

Intermonomer electron transfer between the b hemes of heterodimeric cytochrome bc(1).

The ubihydroquinone:cytochrome c oxidoreductase, or cytochrome bc1, is a central component of respiratory and photosynthetic energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy (ATP) production. The three-dimensional structures of cytochrome bc1 show a homodimeric organization of its three catalytic subunits. The unusual architecture revived the issue of whether the monomers operate independently or function cooperatively during the catalytic cycle of the enzyme. In recent years, different genetic approaches allowed the successful production of heterodimeric cytochrome bc1 variants and evidenced the occurrence of intermonomer electron transfer between the monomers of this enzyme. Here we used a version of the "two-plasmid" genetic system, also described in the preceding paper (DOI: 10.1021/bi400560p), to study a new heterodimeric mutant variant of cytochrome bc1. The strain producing this heterodimeric variant sustained photosynthetic growth of Rhodobacter capsulatus and yielded an active heterodimer. Interestingly, kinetic data showed equilibration of electrons among the four b heme cofactors of the heterodimer, via "reverse" intermonomer electron transfer between the bL hemes. Both inactive homodimeric and active heterodimeric cytochrome bc1 variants were purified to homogeneity from the same cells, and purified samples were subjected to mass spectrometry analyses. The data unequivocally supported the idea that the cytochrome b subunits carried the expected mutations and their associated epitope tags. Implications of these findings on our interpretation of light-activated transient cytochrome b and c redox kinetics and the mechanism of function of a dimeric cytochrome bc1 are discussed with respect to the previously proposed heterodimeric Q cycle model.

Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR.

Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).

Loss of a conserved tyrosine residue of cytochrome b induces reactive oxygen species production by cytochrome bc1.

Production of reactive oxygen species (ROS) induces oxidative damages, decreases cellular energy conversion efficiencies, and induces metabolic diseases in humans. During respiration, cytochrome bc(1) efficiently oxidizes hydroquinone to quinone, but how it performs this reaction without any leak of electrons to O(2) to yield ROS is not understood. Using the bacterial enzyme, here we show that a conserved Tyr residue of the cytochrome b subunit of cytochrome bc(1) is critical for this process. Substitution of this residue with other amino acids decreases cytochrome bc(1) activity and enhances ROS production. Moreover, the Tyr to Cys mutation cross-links together the cytochrome b and iron-sulfur subunits and renders the bacterial enzyme sensitive to O(2) by oxidative disruption of its catalytic [2Fe-2S] cluster. Hence, this Tyr residue is essential in controlling unproductive encounters between O(2) and catalytic intermediates at the quinol oxidation site of cytochrome bc(1) to prevent ROS generation. Remarkably, the same Tyr to Cys mutation is encountered in humans with mitochondrial disorders and in Plasmodium species that are resistant to the anti-malarial drug atovaquone. These findings illustrate the harmful consequences of this mutation in human diseases.

Intermonomer electron transfer between the low-potential b hemes of cytochrome bc1.

Cytochrome (cyt) bc(1) is a structural dimer with its monomers consisting of the Fe-S protein, cyt b, and cyt c(1) subunits. Its three-dimensional architecture depicts it as a symmetrical homodimer, but the mobility of the head domain of the Fe-S protein indicates that the functional enzyme exists in asymmetrical heterodimeric conformations. Here, we report a new genetic system for studying intra- and intermonomer interactions within the cyt bc(1) using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc(1) structural genes carried by two plasmids that are coharbored by a cell without its endogenous enzyme. Our results indicate that coexpressed cyt bc(1) subunits were matured, assorted, and assembled in vivo into homo- and heterodimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of intermonomer electron transfer between the low-potential b hemes of cyt bc(1) was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites of the enzyme. The data demonstrate that active heterodimeric variants, formed of monomers carrying mutations that abolish only one of the two (Q(o) or Q(i)) active sites of each monomer, are produced, and they support photosynthetic growth of R. capsulatus. Detailed analyses of the physicochemical properties of membranes of these mutants, as well as purified homo- and heterodimeric cyt bc(1) preparations, demonstrated that efficient and productive electron transfer occurs between the low-potential b(L) hemes of the monomers in a heterodimeric enzyme. Overall findings are discussed with respect to intra- and intermonomer interactions that take place during the catalytic turnover of cyt bc(1).

Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A.

The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.

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 iron­sulfur protein (ISP) with its Fe2S2 cluster, cyt c1 and cyt b, forming two catalytic domains, the Qo (hydroquinone (QH2) oxidation) and Qi (quinone (Q) reduction) sites. At the Qo 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 Qo 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 Qo site of cyt bc1.

New method for the spin quantitation of [4Fe-4S](+) clusters with S = (3)/(2). Application to the FS0 center of the NarGHI nitrate reductase from Escherichia coli.

In conventional analyses of g approximately 5 signals given by [4Fe-4S](+) clusters with S = 3/2, the effective g values that cannot be measured in the electron paramagnetic resonance (EPR) spectrum are deduced from rhombograms calculated by assuming that the g matrix is isotropic with g(x) = g(y) = g(z) = 2.00. We have shown that when the two low-field peaks corresponding to the Kramers doublets are visible in the spectrum, a new, independent piece of information about the system can be obtained by studying the temperature dependence of the ratio of the area under these peaks. By applying this method to the g approximately 5 signals displayed by NarGHI nitrate reductase, we were able to determine all the parameters of the spin Hamiltonian of FS0 centers with S = 3/2 and to measure accurately their number. Our results indicate that simple analyses based on the assumption of an isotropic g matrix can give rise to very large errors.

Recent advances in cytochrome bc(1): inter monomer electronic communication?

The ubihydroquinone: cytochrome c oxidoreductase, or cytochrome bc(1), is a central component of photosynthetic and respiratory energy transduction pathways in many organisms. It contributes to the generation of membrane potential and proton gradient used for cellular energy production (ATP). The three-dimensional structures of cytochrome bc(1) indicate that its two monomers are intertwined to form a symmetrical homodimer. This unusual architecture raises the issue of whether the monomers operate independently, or function cooperatively during the catalytic cycle of the enzyme. In this review, recent progresses achieved in our understanding of the mechanism of function of dimeric cytochrome bc(1) are presented. New genetic approaches producing heterodimeric enzymes, and emerging insights related to the inter monomer electron transfer between the heme b cofactors of cytochrome bc(1) are described.

Biogenesis of a respiratory complex is orchestrated by a single accessory protein.

The biogenesis of respiratory complexes is a multistep process that requires finely tuned coordination of subunit assembly, metal cofactor insertion, and membrane-anchoring events. The dissimilatory nitrate reductase of the bacterial anaerobic respiratory chain is a membrane-bound heterotrimeric complex nitrate reductase A (NarGHI) carrying no less than eight redox centers. Here, we identified different stable folding assembly intermediates of the nitrate reductase complex and analyzed their redox cofactor contents using electron paramagnetic resonance spectroscopy. Upon the absence of the accessory protein NarJ, a global defect in metal incorporation was revealed. In addition to the molybdenum cofactor, we show that NarJ is required for specific insertion of the proximal iron-sulfur cluster (FS0) within the soluble nitrate reductase (NarGH) catalytic dimer. Further, we establish that NarJ ensures complete maturation of the b-type cytochrome subunit NarI by a proper timing for membrane anchoring of the NarGH complex. Our findings demonstrate that NarJ has a multifunctional role by orchestrating both the maturation and the assembly steps.

High-stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD.

Quinol/nitrate oxidoreductase (NarGHI) is the first enzyme involved in respiratory denitrification in prokaryotes. Although this complex in E. coli is known to operate with both ubi and menaquinones, the location and the number of quinol binding sites remain elusive. NarGHI strongly stabilizes a semiquinone radical located within the dihemic anchor subunit NarI. To identify its location and function, we used a combination of mutagenesis, kinetics, EPR, and ENDOR spectroscopies. For the NarGHIH66Y and NarGHIH187Y mutants lacking the distal heme bD, no EPR signal of the semiquinone was observed. In contrast, a semiquinone was detected in the NarGHIH56Y mutant lacking the proximal heme bP. Its thermodynamic properties and spectroscopic characteristics, as revealed by Q-band EPR and ENDOR spectroscopies, are identical to those observed in the native enzyme. The substitution by Ala of the Lys86 residue close to heme bD, which was previously proposed to be in a quinol oxidation site of NarGHI (QD), also leads to the loss of the EPR signal of the semiquinone, although both hemes are present. Enzymatic assays carried out on the NarGHIK86A mutant reveal that the substitution dramatically reduces the rate of oxidation of both mena and ubiquinol analogues. These observations demonstrate that the semiquinone observed in NarI is strongly associated with heme bD and that Lys86 is required for its stabilization. Overall, our results indicate that the semiquinone is located within the quinol oxidation site QD. Details of the possible binding motif of the semiquinone and mechanistic implications are discussed.

Evidence for an EPR-detectable semiquinone intermediate stabilized in the membrane-bound subunit NarI of nitrate reductase A (NarGHI) from Escherichia coli.

Nitrate reductase A (NRA, NarGHI) is expressed in Escherichia coli by growing the bacterium in anaerobic conditions in the presence of nitrate. This enzyme reduces nitrate to nitrite and uses menaquinol (or ubiquinol) as the electron donor. The location of quinones in the enzyme, their number, and their role in the electron transfer mechanism are still controversial. In this work, we have investigated the spectroscopic and thermodynamic properties of a semiquinone (SQ) in membrane samples of overexpressed E. coli nitrate reductase poised in appropriate redox conditions. This semiquinone is highly stabilized with respect to free semiquinone. The g-values determined from the numerical simulation of its Q-band (35 GHz) EPR spectrum are equal to 2.0061, 2.0051, 2.0023. The midpoint potential of the Q/QH(2) couple is about -100 mV, and the SQ stability constant is about 100 at pH 7.5. The semiquinone EPR signal disappears completely upon addition of the quinol binding site inhibitor 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). A semiquinone radical could also be stabilized in preparations where only the NarI membrane subunit is overexpressed in the absence of the NarGH catalytic dimer. Its thermodynamic and spectroscopic properties show only slight variations with those of the wild-type enzyme. The X-band continuous wave (cw) electron nuclear double resonance (ENDOR) spectra of the radicals display similar proton hyperfine coupling patterns in NarGHI and in NarI, showing that they arise from the same semiquinone species bound to a single site located in the NarI membrane subunit. These results are discussed with regard to the location and the potential function of quinones in the enzyme.