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Copper (Cu)-transporting P1B-type ATPases are ubiquitous metal transporters and crucial for maintaining Cu homeostasis in all domains of life. In bacteria, the P1B-type ATPase CopA is required for Cu-detoxification and exports excess Cu(I) in an ATP-dependent reaction from the cytosol into the periplasm. CopA is a member of the CopA1-type ATPase family and has been biochemically and structurally characterized in detail. In contrast, less is known about members of the CopA2-type ATPase family, which are predicted to transport Cu(I) into the periplasm for cuproprotein maturation. One example is CcoI, which is required for the maturation of cbb3-type cytochrome oxidase (cbb3-Cox) in different species. Here, we reconstituted purified CcoI of Rhodobacter capsulatus into liposomes and determined Cu transport using solid-supported membrane electrophysiology. The data demonstrate ATP-dependent Cu(I) translocation by CcoI, while no transport is observed in the presence of a non-hydrolysable ATP analog. CcoI contains two cytosolically exposed N-terminal metal binding sites (N-MBSs), which are both important, but not essential for Cu delivery to cbb3-Cox. CcoI and cbb3-Cox activity assays in the presence of different Cu concentrations suggest that the glutaredoxin-like N-MBS1 is primarily involved in regulating the ATPase activity of CcoI, while the CopZ-like N-MBS2 is involved in Cu(I) acquisition. The interaction of CcoI with periplasmic Cu chaperones was analyzed by genetically fusing CcoI to the chaperone SenC. The CcoI-SenC fusion protein was fully functional in vivo and sufficient to provide Cu for cbb3-Cox maturation. In summary, our data demonstrate that CcoI provides the link between the cytosolic and periplasmic Cu chaperone networks during cbb3-Cox assembly.

Copper (Cu) is an essential cofactor required for redox enzymes in all domains of life. Because of its toxicity, tightly controlled mechanisms ensure Cu delivery for cuproenzyme biogenesis and simultaneously protect cells against toxic Cu. Many Gram-negative bacteria contain extracytoplasmic multicopper oxidases (MCOs), which are involved in periplasmic Cu detoxification. MCOs are unique cuproenzymes because their catalytic center contains multiple Cu atoms, which are required for the oxidation of Cu1+ to the less toxic Cu2+. Hence, Cu is both substrate and essential cofactor of MCOs. Here, we investigated the maturation of Rhodobacter capsulatus MCO CutO and its role in periplasmic Cu detoxification. A survey of CutO activity of R. capsulatus mutants with known defects in Cu homeostasis and in the maturation of the cuproprotein cbb3-type cytochrome oxidase (cbb3-Cox) was performed. This revealed that CutO activity is largely independent of the Cu-delivery pathway for cbb3-Cox biogenesis, except for the cupric reductase CcoG, which is required for full CutO activity. The most pronounced decrease of CutO activity was observed with strains lacking the cytoplasmic Cu chaperone CopZ, or the Cu-exporting ATPase CopA, indicating that CutO maturation is linked to the CopZ-CopA mediated Cu-detoxification pathway. Our data demonstrate that CutO is important for cellular Cu resistance under both aerobic and anaerobic growth conditions. CutO is encoded in the cutFOG operon, but only CutF, and not CutG, is essential for CutO activity. No CutO activity is detectable when cutF or its putative Cu-binding motif are mutated, suggesting that the cutF product serves as a Cu-binding component required for active CutO production. Bioinformatic analyses of CutF-like proteins support their widespread roles as putative Cu-binding proteins for several Cu-relay pathways. Our overall findings show that the cytoplasmic CopZ-CopA dependent Cu detoxification pathway contributes to providing Cu to CutO maturation, a process that strictly relies on cutF.

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS), and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12 transmembrane helices (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role, and remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His and Cys residues of CcoA we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggested a model linking Cu import for cbb3-Cox biogenesis with a thiol: disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function.ImportanceCopper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here we show that CcoA undergoes a thiol: disulfide oxidoreduction cycle, which is important for its Cu import activity.

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

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Copper (Cu) is an essential trace element for all living organisms and used as cofactor in key enzymes of important biological processes, such as aerobic respiration or superoxide dismutation. However, due to its toxicity, cells have developed elaborate mechanisms for Cu homeostasis, which balance Cu supply for cuproprotein biogenesis with the need to remove excess Cu. This review summarizes our current knowledge on bacterial Cu homeostasis with a focus on Gram-negative bacteria and describes the multiple strategies that bacteria use for uptake, storage and export of Cu. We furthermore describe general mechanistic principles that aid the bacterial response to toxic Cu concentrations and illustrate dedicated Cu relay systems that facilitate Cu delivery for cuproenzyme biogenesis. Progress in understanding how bacteria avoid Cu poisoning while maintaining a certain Cu quota for cell proliferation is of particular importance for microbial pathogens because Cu is utilized by the host immune system for attenuating pathogen survival in host cells.

The respiratory electron transport complexes convey electrons from nutrients to oxygen and generate a proton-motive force used for energy (ATP) production in cells. These enzymes are conserved among organisms, and organized as individual complexes or combined forming large super-complexes (SC). Bacterial electron transport pathways are more branched than those of mitochondria and contain multiple variants of such complexes depending on their growth modes. The Gram-negative species deploy a mitochondrial-like cytochromebc1(Complex III, CIII2), and may have bacteria-specificcbb3-type cytochromecoxidases (Complex IV, CIV) in addition to, or instead of, the canonicalaa3-type CIV. Electron transfer between these complexes is mediated by two different carriers: the soluble cytochromec2which is similar to mitochondrial cytochromecand the membrane-anchored cytochromecywhich is unique to bacteria. Here, we report the first cryo-EM structure of a respiratorybc1-cbb3type SC (CIII2CIV, 5.2Å resolution) and several conformers of native CIII2(3.3Å resolution) from the Gram-negative bacteriumRhodobacter capsulatus. The SC contains all catalytic subunits and cofactors of CIII2and CIV, as well as two extra transmembrane helices attributed to cytochromecyand the assembly factor CcoH. Remarkably, some of the native CIII2are structural heterodimers with different conformations of their [2Fe-2S] cluster-bearing domains. The unresolved cytochromecdomain ofcysuggests that it is mobile, and it interacts with CIII2CIV differently than cytochromec2. Distance requirements for electron transfer suggest that cytochromecyand cytochromec2donate electrons to hemecp1and hemecp2of CIV, respectively. For the first time, the CIII2CIV architecture and its electronic connections establish the structural features of two separate respiratory electron transport pathways (membrane-confined and membrane-external) between its partners in Gram-negative bacteria.

Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have developed sophisticated response mechanisms towards both the lack and the excess of Cu in their environments. In this study, we achieved a global view of Cu-responsive changes in the prokaryotic model organism Rhodobacter capsulatus using label-free quantitative differential proteomics. Semi-aerobically grown cells under heterotrophic conditions in minimal medium (∼0.3 μM Cu) were compared with cells supplemented with either 5 μM Cu or with 5 mM of the Cu-chelator bathocuproine sulfonate. Mass spectrometry based bottom-up proteomics of unfractionated cell lysates identified 2430 of the 3632 putative proteins encoded by the genome, producing a robust proteome dataset for R. capsulatus. Use of biological and technical replicates for each growth condition yielded high reproducibility and reliable quantification for 1926 of the identified proteins. Comparison of cells grown under Cu-excess or Cu-depleted conditions to those grown under minimal Cu-sufficient conditions revealed that 75 proteins exhibited statistically significant (p < 0.05) abundance changes, ranging from 2- to 300-fold. A subset of the highly Cu-responsive proteins was orthogonally probed using molecular genetics, validating that several of them were indeed involved in cellular Cu homeostasis.

PccA and SenC are periplasmic copper chaperones required for the biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox) in Rhodobacter capsulatus at physiological Cu concentrations. However, both proteins are dispensable for cbb3-Cox assembly when the external Cu concentration is high. PccA and SenC bind Cu using Met and His residues and Cys and His residues as ligands, respectively, and both proteins form a complex during cbb3-Cox biogenesis. SenC also interacts directly with cbb3-Cox, as shown by chemical cross-linking. Here we determined the periplasmic concentrations of both proteins in vivo and analyzed their Cu binding stoichiometries and their Cu(I) and Cu(II) binding affinity constants (KD) in vitro. Our data show that both proteins bind a single Cu atom with high affinity. In vitro Cu transfer assays demonstrate Cu transfer both from PccA to SenC and from SenC to PccA at similar levels. We conclude that PccA and SenC constitute a Cu relay system that facilitates Cu delivery to cbb3-Cox.

Combining peroxidase activity-based heme staining (TMBZ/SDS/PAGE) with mass spectrometry analyses (Nano LC-MS/MS) of protein extracts from wild-type and appropriate mutants, we provide evidence that the polychlorinated biphenyl degrader Pseudomonas pseudoalcaligenes KF707 primarily expresses a caa3 -type cytochrome c oxidase (caa3 -Cox) using cytochrome (cyt) c4 as an electron donor in cells grown with biphenyl versus glucose as the sole carbon source. Homology modeling of KF707 caa3 -Cox using the three-dimensional structure of that from Thermus thermophilus highlights multiple similarities and differences between the proton channels in subunit I of the aa3 - and caa3 -Cox of Paracoccus and Thermus spp., respectively. To our knowledge, this is the first report demonstrating the presence of a caa3 -Cox using cyt c4 as an electron donor in a Pseudomonas species.

Copper (Cu)-containing proteins execute essential functions in prokaryotic and eukaryotic cells, but their biogenesis is challenged by high Cu toxicity and the preferential presence of Cu(II) under aerobic conditions, while Cu(I) is the preferred substrate for Cu chaperones and Cu-transport proteins. These proteins form a coordinated network that prevents Cu accumulation, which would lead to toxic effects such as Fenton-like reactions and mismetalation of other metalloproteins. Simultaneously, Cu-transport proteins and Cu chaperones sustain Cu(I) supply for cuproprotein biogenesis and are therefore essential for the biogenesis of Cu-containing proteins. In eukaryotes, Cu(I) is supplied for import and trafficking by cell-surface exposed metalloreductases, but specific cupric reductases have not been identified in bacteria. It was generally assumed that the reducing environment of the bacterial cytoplasm would suffice to provide sufficient Cu(I) for detoxification and cuproprotein synthesis. Here, we identify the proposed cbb(3)-type cytochrome c oxidase (cbb(3)-Cox) assembly factor CcoG as a cupric reductase that binds Cu via conserved cysteine motifs and contains 2 low-potential [4Fe-4S] clusters required for Cu(II) reduction. Deletion of ccoG or mutation of the cysteine residues results in defective cbb(3)-Cox assembly and Cu sensitivity. Furthermore, anaerobically purified CcoG catalyzes Cu(II) but not Fe(III) reduction in vitro using an artificial electron donor. Thus, CcoG is a bacterial cupric reductase and a founding member of a widespread class of enzymes that generate Cu(I) in the bacterial cytosol by using [4Fe-4S] clusters.

The respiratory complexes are organized in supramolecular assemblies called supercomplexes thought to optimize cellular metabolism under physiological and pathological conditions. In this study, we used genetically and biochemically well characterized cells bearing the pathogenic microdeletion m.15,649–15,666 (ΔI300-P305) in MT-CYB gene, to investigate the effects of an assembly-hampered CIII on the re-organization of supercomplexes. First, we found that this mutation also affects the stability of both CI and CIV, and evidences the occurrence of a preferential structural interaction between CI and CIII(2), yielding a small amount of active CI+CIII(2) super-complex. Indeed, a residual CI+CIII combined redox activity, and a low but detectable ATP synthesis driven by CI substrates are detectable, suggesting that the assembly of CIII into the CI+CIII(2) supercomplex mitigates the detrimental effects of MT-CYB deletion. Second, measurements of oxygen consumption and ATP synthesis driven by NADH-linked and FADH(2)-linked substrates alone, or in combination, indicate a common ubiquinone pool for the two respiratory pathways. Finally, we report that prolonged incubation with rotenone enhances the amount of CI and CIII(2), but reduces CIV assembly. Conversely, the antioxidant N-acetylcysteine increases CIII(2) and CIV(2) and partially restores respirasome formation. Accordingly, after NAC treatment, the rate of ATP synthesis increases by two-fold compared with untreated cell, while the succinate level, which is enhanced by the homoplasmic mutation, markedly decreases. Overall, our findings show that fine-tuning the supercomplexes stability improves the energetic efficiency of cells with the MT-CYB microdeletion.

Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the “RfnT-like” CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb3-Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc(1)) 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 Fe(2)S(2) cluster, cyt c(1) and cyt b, forming two catalytic domains, the Q(o) (hydroquinone (QH(2)) oxidation) and Q(i) (quinone (Q) reduction) sites. At the Q(o) site, the electron transfer pathways originating from QH(2) 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 bc(1) 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 bc(1) activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc(1). In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH(2) 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 Fe(2)S(2) 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 QH(2) oxidation at the Q(o) site of cyt bc(1).

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.

Copper (Cu) is an essential micronutrient that functions as a cofactor in several important enzymes, such as respiratory heme-copper oxygen reductases. Yet, Cu is also toxic and therefore cells engage a highly coordinated Cu uptake and delivery system to prevent the accumulation of toxic Cu concentrations. In this study, we analyzed Cu delivery to the cbb3 -type cytochrome c oxidase (cbb3 -Cox) of Rhodobacter capsulatus. We identified the PCuA C-like periplasmic chaperone PccA and analyzed its contribution to cbb3 -Cox assembly. Our data demonstrate that PccA is a Cu-binding protein with a preference for Cu(I), which is required for efficient cbb3 -Cox assembly, in particular, at low Cu concentrations. By using in vivo and in vitro cross-linking, we show that PccA forms a complex with the Sco1-homologue SenC. This complex is stabilized in the absence of the cbb3 -Cox-specific assembly factors CcoGHIS. In cells lacking SenC, the cytoplasmic Cu content is significantly increased, but the simultaneous absence of PccA prevents this Cu accumulation. These data demonstrate that the interplay between PccA and SenC not only is required for Cu delivery during cbb3 -Cox assembly but also regulates Cu homeostasis in R. capsulatus.

Cytochrome c oxidases are members of the heme-copper oxidase superfamily. These enzymes have different subunits, cofactors, and primary electron acceptors, yet they all contain identical heme-copper (CuB) binuclear centers within their catalytic subunits. The uptake and delivery pathways of the CuB atom incorporated into this active site, where oxygen is reduced to water, are not well understood. Our previous work with the facultative phototrophic bacterium Rhodobacter capsulatus indicated that the copper atom needed for the CuB site of cbb3-type cytochrome c oxidase (cbb3-Cox) is imported to the cytoplasm by a major facilitator superfamily-type transporter, CcoA. In this study, a comparative genomic analysis of CcoA orthologs in alphaproteobacterial genomes showed that CcoA is widespread among organisms and frequently co-occurs with cytochrome c oxidases. To define the specificity of CcoA activity, we investigated its function in Rhodobacter sphaeroides, a close relative of R. capsulatus that contains both cbb3- and aa3-Cox. Phenotypic, genetic, and biochemical characterization of mutants lacking CcoA showed that in its absence, or even in the presence of its bypass suppressors, only the production of cbb3-Cox and not that of aa3-Cox was affected. We therefore concluded that CcoA is dedicated solely to cbb3-Cox biogenesis, establishing that distinct copper uptake systems provide the CuB atoms to the catalytic sites of these two similar cytochrome c oxidases. These findings illustrate the large variety of strategies that organisms employ to ensure homeostasis and fine control of copper trafficking and delivery to the target cuproproteins under different physiological conditions., IMPORTANCE The cbb3- and aa3-type cytochrome c oxidases belong to the widespread heme-copper oxidase superfamily. They are membrane-integral cuproproteins that catalyze oxygen reduction to water under hypoxic and normoxic growth conditions. These enzymes diverge in terms of subunit and cofactor composition, yet they all share a conserved heme-copper binuclear site within their catalytic subunit. In this study, we show that the copper atoms of the catalytic center of two similar cytochrome c oxidases from this superfamily are provided by different copper uptake systems during their biogenesis. This finding illustrates different strategies by which organisms fine-tune the trafficking of copper, which is an essential but toxic micronutrient.

The thiol-disulfide oxidoreductase DsbA carries out oxidative folding of extra-cytoplasmic proteins by catalyzing the formation of intramolecular disulfide bonds. It has an important role in various cellular functions, including cell division. The purple non-sulfur bacterium Rhodobacter capsulatus mutants lacking DsbA show severe temperature-sensitive and medium-dependent respiratory growth defects. In the presence of oxygen, at normal growth temperature (35°C), DsbA− mutants form colonies on minimal medium, but they do not grow on enriched medium where cells elongate and lyse. At lower temperatures (i.e., 25°C), cells lacking DsbA grow normally in both minimum and enriched media, however, they do not produce the cbb3-type cytochrome c oxidase (cbb3-Cox) on enriched medium. Availability of chemical oxidants (e.g., Cu2+ or a mixture of cysteine and cystine) in the medium becomes critical for growth and cbb3-Cox production in the absence of DsbA. Indeed, addition of Cu2+ to the enriched medium suppresses, and conversely, omission of Cu2+ from the minimal medium induces, growth and cbb3-Cox defects. Alleviation of these defects by addition of redox-active chemicals indicates that absence of DsbA perturbs cellular redox homeostasis required for the production of an active cbb3-Cox, especially in enriched medium where bioavailable Cu2+ is scarce. This is the first report describing that DsbA activity is required for full respiratory capability of R. capsulatus, and in particular, for proper biogenesis of its cbb3-Cox. We propose that absence of DsbA, besides impairing the maturation of the c-type cytochrome subunits, also affects the incorporation of Cu into the catalytic subunit of cbb3-Cox. Defective high affinity Cu acquisition pathway, which includes the MFS-type Cu importer CcoA, and lower production of the c-type cytochrome subunits lead together to improper assembly and degradation of cbb3-Cox.

A marked stimulation of complex II enzymatic activity was detected in cybrids bearing a homoplasmic MTCYB microdeletion causing disruption of both the activity and the assembly of complex III, but not in cybrids harbouring another MTCYB mutation affecting only the complex III activity. Moreover, complex II stimulation was associated with SDHA subunit tyrosine phosphorylation. Despite the lack of detectable hydrogen peroxide production, up-regulation of the levels of mitochondrial antioxidant defenses revealed a significant redox unbalance. This effect was also supported by the finding that treatment with N-acetylcysteine dampened the complex II stimulation, SDHA subunit tyrosine phosphorylation, and levels of antioxidant enzymes. In the absence of complex III, the cellular amount of succinate, but not fumarate, was markedly increased, indicating that enhanced activity of complex II is hampered due to the blockage of respiratory electron flow. Thus, we propose that complex II phosphorylation and stimulation of its activity represent a molecular mechanism triggered by perturbation of mitochondrial redox homeostasis due to severe dysfunction of respiratory complexes. Depending on the site and nature of the damage, complex II stimulation can either bypass the energetic deficit as an efficient compensatory mechanism, or be ineffectual, leaving cells to rely on glycolysis for survival.

In many Gram-negative bacteria, including Rhodobacter capsulatus, cytochrome c maturation (Ccm) is carried out by a membrane-integral machinery composed of nine proteins (CcmA to I). During this process, the periplasmic thiol-disulfide oxidoreductase DsbA is thought to catalyze the formation of a disulfide bond between the Cys residues at the apocytochrome c heme-binding site (CXXCH). Subsequently, a Ccm-specific thioreductive pathway involving CcmG and CcmH reduces this disulfide bond to allow covalent heme ligation. Currently, the sequence of thioredox reactions occurring between these components and apocytochrome c and the identity of their active Cys residues are unknown. In this work, we first investigated protein-protein interactions among the apocytochrome c, CcmG, and the heme-ligation components CcmF, CcmH, and CcmI. We found that they all interact with each other, forming a CcmFGHI-apocytochrome c complex. Using purified wild-type CcmG, CcmH, and apocytochrome c, as well as their respective Cys mutant variants, we determined the rates of thiol-disulfide exchange reactions between selected pairs of Cys residues from these proteins. We established that CcmG can efficiently reduce the disulfide bond of apocytochrome c and also resolve a mixed disulfide bond formed between apocytochrome c and CcmH. We further show that Cys-45 of CcmH and Cys-34 of apocytochrome c are most likely to form this mixed disulfide bond, which is consistent with the stereo-specificity of the heme-apocytochrome c ligation reaction. We conclude that CcmG confers efficiency, and CcmH ensures stereo-specificity during Ccm and present a comprehensive model for thioreduction reactions that lead to heme-apocytochrome c ligation.

We present a full investigation of ultrafast light-induced events in the membraneous cytochrome bc1 complex by transient absorption spectroscopy. This energy-transducing complex harbors four redox-active components per monomer: heme c1, two 6-coordinate b-hemes and a [2Fe–2S] cluster. Using excitation of these components in different ratios under various excitation conditions, probing in the full visible range and under three well-defined redox conditions, we demonstrate that for all ferrous hemes of the complex photodissociation of axial ligands takes place and that they rebind in 5–7 ps, as in other 6-coordinate heme proteins, including cytoglobin, which is included as a reference in this study. By contrast, the signals are not consistent with photooxidation of the b hemes. This conclusion contrasts with a recent assessment based on a more limited data set. The binding kinetics of internal and external ligands are indicative of a rigid heme environment, consistent with the electron transfer function. We also report, for the first time, photoactivity of the very weakly absorbing iron–sulfur center. This yields the unexpected perspective of studying photochemistry, initiated by excitation of iron–sulfur clusters, in a range of protein complexes.

Correction for ‘Ultrafast photochemistry of the bc1 complex’ by Marten H. Vos et al., Phys. Chem. Chem. Phys., 2017, 19, 6807–6813.

The fission yeast Schizosaccharomyces pombe has been successfully used as a model to gain fundamental knowledge in understanding how eukaryotic cells acquire copper during vegetative growth. These studies have revealed the existence of a heteromeric Ctr4–Ctr5 plasma membrane complex that mediates uptake of copper within the cells. Furthermore, additional studies have led to the identification of one of the first vacuolar copper transporters, Ctr6, as well as the copper-responsive Cuf1 transcription factor. Recent investigations have extended the use of S. pombe to elucidate new roles for copper metabolism in meiotic differentiation. For example, these studies have led to the discovery of Mfc1, which turned out to be the first example of a meiosis-specific copper transporter. Whereas copper-dependent transcriptional regulation of the Ctr family members is under the control of Cuf1 during mitosis or meiosis, meiosis-specific copper transporter Mfc1 is regulated by the recently discovered transactivator Mca1. It is foreseeable that identification of novel meiotic copper-related proteins will serve as stepping stones to unravel fundamental aspects of copper homoeostasis.

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.

Uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive 64Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter., IMPORTANCE Cu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratory cbb3-type cytochrome c oxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated into cbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and for cbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role in cbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer.

Cytochromes (cyts) are ubiquitous heme containing proteins that are key components of energy transduction pathways. They participate in a wide variety of electron transfer reactions, which are essential for cellular processes responsible for chemical energy (ATP) production. The cbb3-type cyt c oxidase (cbb3-Cox) provides an excellent model to study biogenesis of membrane-integral, oligomeric cyt c complexes. Its subunits contain three hemes c, two hemes b and a copper (CuB) atom as cofactors that use distinct insertion processes. In cyts c, heme b is covalently ligated (referred to as heme c) via a complex maturation process that involves in some species up to nine components (Ccm-System I). In addition to the cyts c, many cyt c containing complexes carry other cofactors, and insertion of these cofactors requires additional biogenesis components besides the Ccm-system I. In the case of cbb3-Cox, the mechanisms underlying incorporation of hemes b into the catalytic subunit are not well understood. However, remarkable progress was achieved recently on how the single CuB atom at the catalytic heart of this heme-copper oxidase is acquired. Finally, insertion of the cofactors must be temporally and spatially coordinated with the assembly of the subunits in order to yield a functional cbb3-Cox enzyme. In this chapter, we discuss the biogenesis of cbb3-Cox from the insertion of its catalytic heme-copper (CuB) center and maturation of its c-type cyts to the assembly of its mature subunits, mainly focusing on studies carried out with the anoxygenic phototrophic bacterium Rhodobacter capsulatus.

Cytochromes c are heme proteins that require multiple maturation components, such as heme lyases, for cofactor incorporation. Saccharomyces cerevisiae has two heme lyases that are specific for apocytochromes c (CCHL) or c(1) (CC(1)HL). CCHL can covalently attach heme b groups to apocytochrome c substrates of eukaryotic but not prokaryotic origin. Besides their conserved Cys-Xxx-Xxx-Cys-His heme-binding motifs, the amino-terminal regions of apocytochrome c substrates appear to be important for CCHL function. In this study, we show for the first time that only two amino acid changes in the amino-terminal region of the non-CCHL substrate apocytochrome c(2) from Rhodobacter capsulatus are necessary and sufficient for efficient holocytochrome c formation by CCHL. This finding led us to propose a consensus sequence located at the amino-terminus of apocytochromes c, and critical for substrate recognition and heme ligation by CCHL.

Cytochrome c maturation (Ccm) is a sophisticated post-translational process. It occurs after translocation of apocytochromes c to the p side of energy transducing membranes and forms stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of cysteines at their conserved heme binding sites. In many organisms this process involves up to 10 (CcmABCDEFGHI and CcdA) membrane proteins. One of these proteins is CcmI, which has an N-terminal membrane-embedded domain with two transmembrane helices and a large C-terminal periplasmic domain with protein-protein interaction motifs. Together with CcmF and CcmH, CcmI forms a multisubunit heme ligation complex. How the CcmFHI complex recognizes its apocytochrome c substrates remained unknown. In this study, using Rhodobacter capsulatus apocytochrome c(2) as a Ccm substrate, we demonstrate for the first time that CcmI binds apocytochrome c(2) but not holocytochrome c(2). Mainly the C-terminal portions of both CcmI and apocytochrome c(2) mediate this binding. Other physical interactions via the conserved structural elements in apocytochrome c(2), like the heme ligating cysteines or heme iron axial ligands, are less crucial. Furthermore, we show that the N-terminal domain of CcmI can also weakly bind apocytochrome c(2), but this interaction requires a free thiol group at apocytochrome c(2) heme binding site. We conclude that the CcmI subunit of the CcmFHI complex functions as an apocytochrome c chaperone during the Ccm process used by proteobacteria, archaea, mitochondria of plants and red algae.

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).

The ubihydroquinone:cytochrome c oxidoreductase (cyt bc(1)) contains two catalytically active domains, termed the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites, which are distant from each other by over 30 A. Previously, we have reported that binding of inhibitors to the Q(i) site on one (n) side of the energy-transducing membrane changes the local environment of the iron-sulfur (Fe/S) protein subunit residing in the Q(o) site on the other (p) side of the lipid bilayer [Cooley, J. W., Ohnishi, T., and Daldal, F. (2005) Biochemistry 44, 10520-10532]. These findings best fit a model whereby the Q(o) and Q(i) sites of the cyt bc(1) are actively coupled in spite of their distant locations. Because the Fe/S protein of the cyt bc(1) undergoes a large-scale (macro) domain movement during catalysis, we examined various macromobility-defective Fe/S subunit mutants to assess the role of this motion on the coupling of the active sites and also during the multiple turnovers of the enzyme. By monitoring the changing environments of the Fe/S protein [2Fe-2S] cluster upon addition of Q(i) site inhibitors in selected mutants, we found that the Q(o)-Q(i) site interactions manifest differently depending on the ability of the Fe/S protein to move between the cytochrome b and cytochrome c(1) subunits of the enzyme. In the presence of antimycin A, an immobile Fe/S protein mutant exhibited no changes in its EPR spectra. In contrast, mobility-restricted mutants showed striking alterations in the EPR line shapes and revealed two discrete subpopulations in respect to the [2Fe-2S] cluster environments at the Q(o) site. These findings led us to conclude that the mobility of the Fe/S protein is involved in its response to the occupancy of the Q(i) site by different molecules. We propose that the heterogeneity seen might reflect the distinct responses of the two Fe/S proteins at the Q(o) sites of the dimeric enzyme upon the occupancy of the Q(i) sites and discuss it in terms of the function of the dimeric cyt bc(1) during its multiple turnovers.

Extensive molecular modeling based on crystallographic data was used to aid the design of synthetic analogues of the fungicidal naturally occurring respiration inhibitors crocacins A and D, and an inhibitor binding model to the mammalian cytochrome bc(1) complex was constructed. Simplified analogues were made which showed high activity in a mitochondrial beef heart respiration assay, and which were also active against certain plant pathogens in glasshouse tests. A crystal structure was obtained of an analogue of crocacin D bound to the chicken heart cytochrome bc(1) complex, which validated the binding model and which confirmed that the crocacins are a new class of inhibitor of the cytochrome bc(1) complex.

Cytochrome c maturation (Ccm) is a post-translational and post-export protein modification process that involves ten (CcmABCDEFGHI and CcdA or DsbD) components in most Gram-negative bacteria. The absence of any of these components abolishes the ability of cells to form cytochrome c, leading in the case of Rhodobacter capsulatus to the loss of photosynthetic proficiency and respiratory cytochrome oxidase activity. Based on earlier molecular genetic studies, we inferred that R. capsulatus CcmF, CcmH, and CcmI interact with each other to perform heme-apocytochrome c ligation. Here, using functional epitope-tagged derivatives of these components coproduced in appropriate mutant strains, we determined protein-protein interactions between them in detergent-dispersed membranes. Reciprocal affinity purification as well as tandem size exclusion and affinity chromatography analyses provided the first biochemical evidence that CcmF, CcmH, and CcmI associate stably with each other, indicating that these Ccm components form a membrane-integral complex. Under the conditions used, the CcmFHI complex does not contain CcmG, suggesting that the latter thio-reduction component is not always associated with the heme ligation components. The findings are discussed with respect to defining the obligatory components of a minimalistic heme-apocytochrome c ligation complex in R. capsulatus.

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.

Cytochrome cbb 3 -type oxidases are members of the heme copper oxidase superfamily and are composed of four subunits. CcoN contains the heme b -Cu B binuclear center where oxygen is reduced, while CcoP and CcoO are membrane-bound c -type cytochromes thought to channel electrons from the donor cytochrome into the binuclear center. Like many other bacterial members of this superfamily, the cytochrome cbb 3 -type oxidase contains a fourth, non-cofactor-containing subunit, which is termed CcoQ. In the present study, we analyzed the role of CcoQ on the stability and activity of Rhodobacter capsulatus cbb 3 -type oxidase. Our data showed that CcoQ is a single-spanning membrane protein with a N out -C in topology. In the absence of CcoQ, cbb 3 -type oxidase activity is significantly reduced, irrespective of the growth conditions. Blue native polyacrylamide gel electrophoresis analyses revealed that the lack of CcoQ specifically impaired the stable recruitment of CcoP into the cbb 3 -type oxidase complex. This suggested a specific CcoQ-CcoP interaction, which was confirmed by chemical cross-linking. Collectively, our data demonstrated that in R. capsulatus CcoQ was required for optimal cbb 3 -type oxidase activity because it stabilized the interaction of CcoP with the CcoNO core complex, leading subsequently to the formation of the active 230-kDa cbb 3 -type oxidase complex.

Photosynthetic (Ps) growth of purple non-sulfur bacteria such as Rhodobacter capsulatus depends on the cyclic electron transfer (ET) between the ubihydroquinone (QH(2)): cytochrome (cyt) c oxidoreductases (cyt bc(1) complex), and the photochemical reaction centers (RC), mediated by either a membrane-bound (cyt c(y)) or a freely diffusible (cyt c(2)) electron carrier. Previously, we constructed a functional cyt bc(1)-c(y) fusion complex that supported Ps growth solely relying on membrane-confined ET (Lee, D.-W., Ozturk, Y., Mamedova, A., Osyczka, A., Cooley, J. W., and Daldal, F. (2006) Biochim. Biophys. Acta 1757, 346 352). In this work, we further characterized this cyt bc1-cy fusion complex, and used its derivatives with shorter cyt c(y) linkers as "molecular rulers" to probe the distances separating the Ps components. Comparison of the physicochemical properties of both membrane-embedded and purified cyt bc(1)-c(y) fusion complexes established that these enzymes were matured and assembled properly. Light-activated, time-resolved kinetic spectroscopy analyses revealed that their variants with shorter cyt c(y) linkers exhibited fast, native-like ET rates to the RC via the cyt bc(1). However, shortening the length of the cyt (c)y linker decreased drastically this electronic coupling between the cyt bc(1)-c(y) fusion complexes and the RC, thereby limiting Ps growth. The shortest and still functional cyt c(y) linker was about 45 amino acids long, showing that the minimal distance allowed between the cyt bc(1)-c(y) fusion complexes and the RC and their surrounding light harvesting proteins was very short. These findings support the notion that membrane-bound Ps components form large, active structural complexes that are "hardwired" for cyclic ET.

The c-type cytochromes are electron transfer proteins involved in energy transduction. They have heme-binding (CXXCH) sites that covalently ligate heme b via thioether bonds and are classified into different classes based on their protein folds and the locations and properties of their cofactors. Rhodobacter capsulatus produces various c-type cytochromes using the cytochrome c maturation (Ccm) System I, formed from the CcmABCDEFGHI proteins. CcmI, a component of the heme ligation complex CcmFHI, interacts with the heme-handling protein CcmE and chaperones apocytochrome c2 by binding its C-terminal helix. Whether CcmI also chaperones other c-type apocytochromes, and the effects of heme on these interactions were unknown previously. Here, we purified different classes of soluble and membrane-bound c-type apocytochromes (class I, c2 and c1, and class II c') and investigated their interactions with CcmI and apoCcmE. We report that, in the absence of heme, CcmI and apoCcmE recognized different classes of c-type apocytochromes with different affinities (nM to μM KD values). When present, heme induced conformational changes in class I apocytochromes (e.g. c2) and decreased significantly their high affinity for CcmI. Knowing that CcmI does not interact with mature cytochrome c2 and that heme converts apocytochrome c2 into its b-type derivative, these findings indicate that CcmI holds the class I apocytochromes (e.g. c2) tightly until their noncovalent heme-containing b-type cytochrome-like intermediates are formed. We propose that these intermediates are subsequently converted into mature cytochromes following the covalent ligation of heme via the remaining components of the Ccm complex.

In Rhodobacter capsulatus excess reducing equivalents generated by organic acid oxidation is consumed to reduce protons into hydrogen by the activity of nitrogenase. Nitrogenase serves as a redox-balancing tool and is activated by the RegB/RegA global regulatory system during photosynthetic growth. The terminal cytochrome cbb 3 oxidase and the redox state of the cyclic photosynthetic electron transfer chain serve redox signaling to the RegB/RegA regulatory systems in Rhodobacter. In this study, hydrogen production of various R. capsulatus strains harboring the genetically modified electron carrier cytochromes or lacking the cyt cbb 3 oxidase or the quinol oxidase were compared with the wild type. The results indicated that hydrogen production of mutant strains with modified electron carrier cytochromes decreased 3- to 4-fold, but the rate of hydrogen production increased significantly in a cbb 3 - mutant. Moreover, hydrogen production efficiency of various R. capsulatus strains further increased by inactivation of uptake hydrogenase genes.

Comparison and identification of mitochondrial matrix proteins from wild-type and cysteine desulfurase-defective (nfs1-14, carrying a hypomorphic allele of NFS1) yeast strains, using two-dimensional gel electrophoresis coupled to mass spectrometry analyses, revealed large changes in the amounts of various proteins. Protein spots that were specifically increased in the nfs1-14 mutant included subunits of lipoamide-containing enzyme complexes: Kgd2, Lat1, and Gcv3, subunits of the mitochondrial alpha-ketoglutarate dehydrogenase, pyruvate dehydrogenase, and glycine cleavage system complexes, respectively. Moreover the increased protein spots corresponded to lipoamide-deficient forms in the nfs1-14 mutant. The increased proteins migrated as separate, cathode-shifted spots, consistent with gain of a lysine charge due to lack of lipoamide addition. Lack of lipoylation of these proteins was further validated using an antibody specific for lipoamide-containing proteins. In addition, this antibody revealed a fourth lipoamide-containing protein, probably corresponding to the E2 component of the branched-chain keto acid dehydrogenase complex. Like the lipoamide-containing forms of Kgd2, Lat1, and Gcv3, this protein also showed decreased lipoic acid reactivity in the nfs1-14 mutant. Cysteine desulfurases, such as yeast NFS1, are required for sulfur addition to iron-sulfur clusters and other sulfur-requiring processes. The results demonstrate that Nfs1 protein is required for the proper post-translational modification of the lipoamide-containing mitochondrial subproteome in yeast and pave the road toward a thorough understanding of its precise role in lipoic acid synthesis.

Gram-negative bacteria like Rhodobacter capsulatus use intertwined pathways to carry out the posttranslational maturation of c -type cytochromes (Cyts). This periplasmic process requires at least 10 essential components for apo-Cyt c chaperoning, thio-oxidoreduction, and the delivery of heme and its covalent ligation. One of these components, CcmI (also called CycH), is thought to act as an apo-Cyt c chaperone. In R. capsulatus , CcmI-null mutants are unable to produce c -type Cyts and thus sustain photosynthetic (Ps) growth. Previously, we have shown that overproduction of the putative heme ligation components CcmF and CcmH Rc (also called Ccl1 and Ccl2) can partially bypass the function of CcmI on minimal, but not on enriched, media. Here, we demonstrate that either additional overproduction of CcmG (also called HelX) or hyperproduction of CcmF-CcmH Rc is needed to completely overcome the role of CcmI during the biogenesis of c -type Cyts on both minimal and enriched media. These findings indicate that, in the absence of CcmI, interactions between the heme ligation and thioreduction pathways become restricted for sufficient Cyt c production. We therefore suggest that CcmI, along with its apo-Cyt chaperoning function, is also critical for the efficacy of holo-Cyt c formation, possibly via its close interactions with other components performing the final heme ligation steps during Cyt c biogenesis.

Escherichia coli contains a major phosphofructokinase isoenzyme, phosphofructokinase 1, which is allosteric, and a minor isoenzyme, phosphofructokinase 2. The pfkB1 mutation is known to increase the amount of phosphofructokinase 2 and allow growth on sugars of mutants lacking phosphofructokinase 1; it does not affect growth on substances such as glycerol or lactate (i.e., 'gluconeogenic growth'). However, gluconeogenic growth is markedly impaired in strains with a different allele, pfkB1*. We show here that strains with pfkB1* contain an altered form of phosphofructokinase 2, called phosphofructokinase 2*, which has been purified. Phosphofructokinase 2* is cold labile and has slightly different kinetic characteristics from phosphofructokinase 2, which include being less sensitive to inhibition by fructose 1,6-bisphosphate. The Km for fructose 6-phosphate is low (about 5 X 10(-5) M) in both phosphofructokinase 2 and phosphofructokinase 2*. However, in strains lacking phosphofructokinase 1, a high level of phosphofructokinase 2 is associated with unusually high concentrations of hexose monophosphates during growth on glucose, while a strain with phosphofructokinase 2* instead of phosphofructokinase 2 grows more rapidly on glucose and contains lower levels of hexose monophosphates. In gluconeogenic conditions, by contrast, hexose monophosphate levels are normal in phosphofructokinase 2 strains, while the impaired growth of phosphofructokinase 2* strains is associated with high levels of fructose 2,6-bisphosphate and very low levels of hexose monophosphates. These results show that phosphofructokinase 2, as studied in vitro, should no longer be regarded as a 'non-allosteric' protein, a conclusion also reached by Kotlarz and Buc on the basis of different types of experiments [Eur. J. Biochem. 117, 569-574 (1981)]. The fact that mutational alteration of phosphofructokinase 2 allows more rapid growth on glucose but severely impairs gluconeogenic growth is an indication of the significance of the regulation in vivo. The more rapid growth of the mutant on glucose might be explained on the basis of decreased sensitivity to an inhibitor (possibly, but not necessarily, fructose 1,6-bisphosphate), although other models are possible. A variety of speculations are offered as to the mechanism of gluconeogenic impairment.

Reversibility is a common theme in respiratory and photosynthetic systems that couple electron transfer with a transmembrane proton gradient driving ATP production. This includes the intensely studied cytochrome bc1, which catalyses electron transfer between quinone and cytochrome c. To understand how efficient reversible energy coupling works, here we have progressively inactivated individual cofactors comprising cytochrome bc1. We have resolved millisecond reversibility in all electron-tunnelling steps and coupled proton exchanges, including charge-separating hydroquinone-quinone catalysis at the Q(o) site, which shows that redox equilibria are relevant on a catalytic timescale. Such rapid reversibility renders popular models based on a semiquinone in Q(o) site catalysis prone to short-circuit failure. Two mechanisms allow reversible function and safely relegate short-circuits to long-distance electron tunnelling on a timescale of seconds: conformational gating of semiquinone for both forward and reverse electron transfer, or concerted two-electron quinone redox chemistry that avoids the semiquinone intermediate altogether.

The ubihydroquinone:cytochrome c oxidoreductase (also called complex III, or bc (1) complex), is a multi subunit enzyme encountered in a very broad variety of organisms including uni- and multi-cellular eukaryotes, plants (in their mitochondria) and bacteria. Most bacteria and mitochondria harbor various forms of the bc (1) complex, while plant and algal chloroplasts as well as cyanobacteria contain a homologous protein complex called plastohydroquinone:plastocyanin oxidoreductase or b (6) f complex. Together, these enzyme complexes constitute the superfamily of the bc complexes. Depending on the physiology of the organisms, they often play critical roles in respiratory and photosynthetic electron transfer events, and always contribute to the generation of the proton motive force subsequently used by the ATP synthase. Primarily, this review is focused on comparing the 'mitochondrial-type' bc (1) complex and the 'chloroplast-type' b (6) f complex both in terms of structure and function. Specifically, subunit composition, cofactor content and assembly, inhibitor sensitivity, proton pumping, concerted electron transfer and Fe-S subunit large-scale domain movement of these complexes are discussed. This is a timely undertaking in light of the structural information that is emerging for the b (6) f complex.

The ubihydroquinone:cytochrome (cyt) c oxidoreductase, or bc(1) complex, and its homologue the b(6)f complex are key components of respiratory and photosynthetic electron transport chains as they contribute to the generation of an electrochemical gradient used by the ATP synthase to produce ATP. The bc(1) complex has two catalytic domains, ubihydroquinone oxidation (Q(o)) and ubiquinone reduction (Q(i)) sites, that are located on each side of the membrane. The key to the energetic efficiency of this enzyme relies upon the occurrence of a unique electron bifurcation reaction at its Q(o) site. Recently, several lines of evidence have converged to establish that in the bc(1) complex the extrinsic domain of the Fe-S subunit that contains a [2Fe2S] metal cluster moves during catalysis to shuttle electrons between the Q(o) site and c(1) heme. While this step is required for electron bifurcation, available data also suggest that the movement might be controlled to ensure maximal energetic efficiency [Darrouzet et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 4567-4572]. To gain insight into the plausible control mechanism, we used a biochemical genetic approach to define the different regions of the bc(1) complex that might interact with each other. Previously, we found that a mutation located at position L286 of the ef loop of Rhodobacter capsulatus cyt b could alleviate movement impairment resulting from a mutation in the hinge region, linking the [2Fe2S] cluster domain to the membrane anchor of the Fe-S subunit. Here we report that various substitutions at position 288 on the opposite side of the ef loop also impair Q(o) site catalysis. In particular, we note that while most of the substitutions affect only QH(2) oxidation, yet others like T288S also hinder the rate of the movement of the Fe-S subunit. Thus, position 288 of cyt b appears to be important for both the QH(2) oxidation and the movement of the Fe-S subunit. Moreover, we found that, upon substitution of T288 by other amino acids, additional compensatory mutations located at the [2Fe2S] cluster or the hinge domains of the Fe-S subunit, or on the cd loop of cyt b, arise readily to alleviate these defects. These studies indicate that intimate protein-protein interactions occur between cyt b and the Fe-S subunits to sustain fast movement and efficient QH(2) oxidation and highlight the critical dual role the ef loop of cyt b to fine-tune the docking and movement of the Fe-S subunit during Q(o) site catalysis.

Recent kinetics experiments using mutants of thebc 1 complex (ubihydroquinone-cytochromec oxidoreductase) iron-sulfur subunit with modified hinge regions have revealed the crucial role played by the large scale movement of its [2Fe-2S] cluster domain during the activity of this enzyme. In particular, one of these mutants (+1Ala) with an insertion of one alanine residue in the hinge region is partially deficient in performing this movement. We found that this defect can be overcome by the appearance of a second mutation substituting the leucine at position 286 in the ef loop of cytochromeb with a phenylalanine. Detailed studies of these mutants and their derivatives revealed that the ef loop acts as a barrier that needs to be crossed for multiple turnovers of the enzyme but not for a single turnover ubihydroquinone oxidation site catalysis. These findings indicate that the movement of the iron-sulfur subunit is composed of two discrete parts: a “micro-movement” at the cytochrome b interface, during which the [2Fe-2S] cluster interacts with ubihydroquinone oxidation site occupants and catalyzes ubihydroquinone oxidation, and a “macro-movement,” during which the cluster domain swings away from cytochrome binterface, crosses the ef loop, and reaches a position close to cytochrome c 1 heme, to which it ultimately transfers an electron.

Recent crystallographic and kinetic data have revealed the crucial role of the large scale domain movement of the iron-sulfur subunit [2Fe-2S] cluster domain during the ubihydroquinone oxidation reaction catalyzed by the cytochrome bc(1) complex. Previously, the electron paramagnetic resonance signature of the [2Fe-2S] cluster and its redox midpoint potential (E(m)) value have been used extensively to characterize the interactions of the [2Fe-2S] cluster with the occupants of the ubihydroquinone oxidation (Q(o)) catalytic site. In this work we analyze these interactions in various iron-sulfur subunit mutants that carry mutations in its flexible hinge region. We show that the E(m) increases of the iron-sulfur subunit [2Fe-2S] cluster induced either by these mutations or by the addition of stigmatellin do not act synergistically. Moreover, the E(m) increases disappear in the presence of class I inhibitors like myxothiazol. Because various inhibitors are known to affect the location of the iron-sulfur subunit cluster domain, the measured E(m) value of the [2Fe-2S] cluster therefore reflects its equilibrium position in the Q(o) site. We also demonstrate the existence in this site of a location where the E(m) of the cluster is increased by about 150 mV and discuss its possible implications in term of Q(o) site catalysis and energetics.

Cytochromes c are ubiquitous heme proteins that are found in most living organisms and are essential for various energy production pathways as well as other cellular processes. Their biosynthesis relies on a complex post-translational process, called cytochrome c biogenesis, responsible for the formation of stereo-specific thioether bonds between the vinyl groups of heme b (protoporphyrin IX-Fe) and the thiol groups of apocytochromes c heme-binding site (C1XXC2H) cysteine residues. In some organisms this process involves up to nine (CcmABCDEFGHI) membrane proteins working together to achieve heme ligation, designated the Cytochrome c maturation (Ccm)-System I. Here, we review recent findings related to the Ccm-System I found in bacteria, archaea and plant mitochondria, with an emphasis on protein interactions between the Ccm components and their substrates (apocytochrome c and heme). We discuss the possibility that the Ccm proteins may form a multi subunit supercomplex (dubbed “Ccm machine”), and based on the currently available data, we present an updated version of a mechanistic model for Ccm. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome c oxidase as well as many bacterial homologues. Members of the C family, also called the cbb 3 -type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the K C channel) present in the cbb 3 -type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the K C channel of the cbb 3 -type oxygen reductase from Rhodobacter capsulatus were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the K C channel.