Cytochrome (cyt) bc1 is a structural dimer with its monomers consisting of the Fe/S protein, cyt b and cyt c1 subunits. Its three-dimensional architecture depicts it as a symmetrical homo-dimer, but the mobility of the head domain of the Fe/S protein indicates that the functional enzyme undergoes asymmetrical hetero-dimeric conformations. Here, we report a new genetic system for studying intra and inter-monomer interactions within the cyt bc1 using the facultative phototrophic bacterium Rhodobacter capsulatus. The system involves two different sets of independently expressed cyt bc1 structural genes carried by two plasmids that are co-harbored by a cell without its endogenous enzyme. Our results indicate that co-expressed cyt bc1 subunits are matured, assorted and assembled in vivo into homo- and hetero-dimeric enzymes that can bear different mutations in each monomer. Using the system, the occurrence of inter-monomer electron transfer between the low potential b hemes of cyt bc1 was probed by choosing mutations that perturb electron transfer at the hydroquinone oxidation (Qo) and quinone reduction (Qi) sites of the enzyme. The data demonstrate that active hetero-dimeric variants, formed of monomers carrying mutations that abolish only one of the two (Qo or Qi) 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 hetero-dimeric cyt bc1 preparations, demonstrate that efficient and productive electron transfer occurs between the low potential hemes bL of the monomers in a hetero-dimeric enzyme. Overall findings are discussed with respect to intra- and inter-monomer interactions that occur during the catalytic turnover of cyt bc1.

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.All biological systems require the biogenesis of functional respiratory or photosynthetic complexes for their viability. In bacteria, bioenergetic electron transfer chains are associated to the inner membrane. Biogenesis of these complexes is an intricate process that requires several steps such as the synthesis of the different subunits, their assembly, the incorporation of various types of metal or organic cofactors, and the anchoring of the complex to the membrane. In the case of exported metalloproteins, the assembly and cofactor incorporation steps need to be accomplished prior to translocation of the inner membrane via the twin arginine translocase apparatus (1-3). Importantly, accessory proteins are often involved in biogenesis of metalloproteins (4 -9). Although it is most likely that all these events occur in a coordinate fashion to yield a final functional multimeric metalloprotein, information about how this coordination is performed is scarce.The well studied and characterized Escherichia coli dissimilatory quinol-nitrate oxidoreductase of the anaerobic respiratory chain, referred to as the nitrate reductase A (NarGHI) 4 (10, 11), can be considered as a suitable model for deciphering the biogenesis pathway of multimeric metalloproteins. NarGHI is a non-exported membrane-bound respiratory complex composed of three subunits that bind eight redox centers: (i) a catalytic subunit (NarG) containing a molybdenum-bis-molybdopterin guanine dinucleotide cofactor (Moco) and a proximal [4Fe-4S] cluster (FS0) (12, 13), (ii) an electron transfer subunit (NarH) carrying one [3Fe-4S] cluster (FS4) and three [4Fe-4S] clusters (FS1 to FS3) (14, 15), and (iii) a quinol-oxidizing membrane-bound subunit (NarI) containing two b-type hemes (b D and b P ) (11,16,17). The NarJ protein encoded by the narGHJI operon plays an essential role in nitrate reductase activity, enabling Moco insertion...

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.bioenergetics | EPR spectroscopy | metalloprotein | molybdenum

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.

In the absence of oxygen and in the presence of nitrate, Escherichia coli induces the production of two energy-converting enzymes: formate dehydrogenase-N (FdnGHI) and dissimilatory nitrate reductase A (NarGHI).3 These two complexes cooperate in generating a proton motive force through the redox loop mechanism as originally envisaged by Peter Mitchell in his chemiosmotic hypothesis (1). The separation of positive and negative charges across the cytoplasmic membrane is achieved through electron transfer from the formate oxidation site in the periplasm to the nitrate reduction site located on the cytoplasmic space with mena-or ubiquinone/quinol cycling between them (2, 3). The heterotrimeric NarGHI complex is composed of (i) a nitrate-reducing subunit NarG containing a Mo-bis-MGD cofactor (Moco) and a [4Fe-4S] cluster FeS0 with unusual His coordination (4, 5), (ii) an electron transfer subunit NarH carrying four FeS clusters (6), and (iii) a membrane anchor subunit NarI containing two b-type hemes termed b D and b P to indicate their distal and proximal positions with respect to the nitratereducing site (7-9). These prosthetic groups define an electron transfer pathway from a periplasmically oriented quinol oxidation site (Q D ) to a cytoplasmically oriented nitrate reduction site.Although considerable research has been devoted to nitrate reductase functioning, only partial information has been gained about the number, the structure, and the specificity of quinol binding sites within NarGHI. For instance, some studies have reported on mena-and ubiquinol analogue binding (10 -20), whereas the molecular details of the interaction between natural quinols and NarGHI remain to be established.Despite the absence of bound quinones in the available high resolution structures of NarGHI (7,20), the crystal structure of the enzyme in complex with pentachlorophenol (PCP), an inhibitor of the quinol oxidase activity, has been determined (20). Based on mutagenesis data, biochemical analyses, and molecular modeling, a model of the Q D quinol binding site has been proposed (20). In this working model, a quinone carbonyl group interacts with the protein via a hydrogen bond to a histidine residue (His-66), which is one of the axial ligands of heme b D . Molecular modeling of a menaquinone in the PCP binding site suggested that the opposite carbonyl group could form a hydrogen bond to . Additionally, an elongated

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.

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.In most organisms, the ubiquinol:cytochrome c oxidoreductase (cytochrome bc 1 or complex III) is a central enzyme for ATP production through oxidative phosphorylation that relies on the proton motive force (⌬H ϩ ) generated by the respiratory chain (1). Production of reactive oxygen species (ROS) leads oxidative damages of cellular components and in eukaryotes induces apoptosis (2, 3). Most cellular ROS are thought to emanate from the respiratory NADH:dehydrogenase (i.e. complex I) and cytochrome bc 1 (see Fig. 1a for Rhodobacter capsulatus structure) under compromising physiological conditions (4, 5). Cells use antioxidant enzymes (e.g. superoxide dismutase or glutathione peroxidase) to prevent oxidative damages, but upon extensive ROS generation, harmful damage occurs. Indeed, cellular redox homeostasis, regulated by the rate of electron flow through the respiratory chain and O 2 availability, is tightly coupled with the global metabolism (4, 5). For example, recent studies show that cytochrome bc 1 is involved in stabilization and activation of hypoxia-induced factors like HIF-1␣ by mitochondria-generated ROS under hypoxic conditions (6, 7).Mitochondrial DNA mutations are known causes of clinical syndromes (e.g. LHON, Pearson syndrome, and exercise intolerance) or provide predisposition for inherited and common diseases (e.g. aging, cardiomyopathy, cancer, diabetes, and neurodegenerative diseases) (8, 9). Mutations in mitochondrial or nuclear DNAs associated with mitochondrial fission and fusion (10), ascribed to ROS generation, lead to progressive dysfunction of mitochondria and loss of energy efficiency (11). For example, the Tyr to Cys mutation at position 278 (position 302 in R. capsulatus; see Fig. 1b) of cytochrome b of human mitochondrial cytochrome bc 1...

In this minireview, 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 (Y302 in R. capsulatus and Y278 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.