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Cytochrome c oxidase is the terminal complex of the respiratory chains in the mitochondria of nearly all eu-karyotes. It catalyzes the reduction of molecular O-2 to water using electrons from the respiratory chain, delivered via cytochrome c on the external surface of the inner mitochondrial membrane. The protons required for water formation are taken from the matrix side of the membrane, making catalysis vectorial. This vectorial feature is further enhanced by the fact that the redox catalysis is coupled to the translocation of protons from the inside to the outside of the inner mitochondrial membrane. We are dealing with a molecular machine that converts redox free energy into a protonmotive force (pmf). Here, we review the current extensive knowledge of the structural changes in the active heme-copper site that accompany catalysis, based on a large variety of time-resolved spectroscopic experiments, X-ray and cryoEM structures, and advanced computational chemistry.

Breaking down oxygen Molecular oxygen (O 2 ) is the terminal oxidant for respiration in mitochondria and many bacteria. Within membrane-bound heme–copper oxidases, a controlled, four-electron reduction of O 2 to water is coupled to pumping of protons across the membrane that can be used, among other outcomes, to generate adenosine triphosphate. Studying cytochrome bo 3 ubiquinol oxidase, Jose et al . investigated the key P M intermediate, which forms after O–O bond cleavage and precedes proton pumping, using magnetic circular dichroism spectroscopy. The authors observed features demonstrating that P M is a three-spin system, which is consistent with a consensus model including an iron(IV)-oxo species, copper(II) ion, and tyrosyl radical. These results provide an important validation of the O–O cleavage mechanism and open the door to understanding the proton pumping step. —MAF

The effect of Zn2+ on the P-side of proteoliposomes containing membrane-incorporated Rhodobacter sphaeroides cytochrome c oxidase was investigated by the time-resolved electrometrics following a single electron injection into the enzyme prepared in the F state. The wild-type enzyme was examined along with the two mutants, N139D and D132N. All obtained data indicate that the primary effect of Zn2+ added from the P-side of the membrane is slowing of the pumped proton release from the proton loading site (PLS) to the bulk aqueous phase on the P-side of the membrane. The results strongly suggest the presence of two pathways by which the pumped proton can exit the protein from the PLS and of two separate binding sites for Zn2+. A model is presented to explain the influence of Zn2+ on the kinetics of membrane-potential generation by the wild-type COX, as well as by the N139D and D132N mutants.

Cis-prenyltransferases such as undecaprenyl diphosphate synthase (UPPS) and decaprenyl diphosphate synthase (DPPS) are essential enzymes in bacteria and are involved in cell wall biosynthesis. UPPS and DPPS are absent in the human genome, so they are of interest as targets for antibiotic development. Here, we screened a library of 750 compounds from National Cancer Institute Diversity Set V for the inhibition of Mycobacterium tuberculosis DPPS and found 17 hits, and then IC50s were determined using dose-response curves. Compounds were tested for growth inhibition against a panel of bacteria, for in vivo activity in a Staphylococcus aureus/Caenorhabditis elegans model, and for mammalian cell toxicity. The most active DPPS inhibitor was the dicarboxylic acid redoxal (compound 10), which also inhibited undecaprenyl diphosphate synthase (UPPS) as well as farnesyl diphosphate synthase. 10 was active against S. aureus, Clostridiodes difficile, Bacillus anthracis Sterne, and Bacillus subtilis, and there was a 3.4-fold increase in IC50 on addition of a rescue agent, undecaprenyl monophosphate. We found that 10 was also a weak protonophore uncoupler, leading to the idea that it targets both isoprenoid biosynthesis and the proton motive force. In an S. aureus/C. elegans in vivo model, 10 reduced the S. aureus burden 3 times more effectively than did ampicillin.

The heme‑copper oxidoreductase (HCO) superfamily is a large superfamily of terminal respiratory enzymes that are widely distributed across the three domains of life. The superfamily includes biochemically diverse oxygen reductases and nitric oxide reductases that are pivotal in the pathways of aerobic respiration and denitrification. The adaptation of HCOs to use quinol as the electron donor instead of cytochrome c has significant implication for the respiratory flexibility and energetic efficiency of the respiratory chains that include them. In this work, we explore the adaptation of this scaffold to two different electron donors, cytochromes c and quinols, with extensive sequence analysis of these enzymes from publicly available datasets. Our work shows that quinol oxidation evolved independently within the HCO superfamily at least seven times. Enzymes from only two of these independently evolved clades have been biochemically well-characterized. Combining structural modeling with sequence analysis, we identify putative quinol binding sites in each of the novel quinol oxidases. Our analysis of experimental and modeling data suggests that the quinol binding site appears to have evolved at the same structural position within the scaffold more than once.

Nitrogen is an essential element for life, with the availability of fixed nitrogen limiting productivity in many ecosystems. The return of oxidized nitrogen species to the atmospheric N2 pool is predominately catalyzed by microbial denitrification (NO3- → NO2- → NO → N2O → N2)1. Incomplete denitrification can produce N2O as a terminal product, leading to an increase in atmospheric N2O, a potent greenhouse and ozone-depleting gas2. The production of N2O is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) superfamily3. Here we use phylogenomics to identify a number of previously uncharacterized HCO families and propose that many of them (eNOR, sNOR, gNOR, and nNOR) perform nitric oxide reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple nitric oxide reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from Rhodothermus marinus, verifying that it performs nitric oxide reduction both in vitro and in vivo. These newly identified NORs exhibit broad phylogenetic and environmental distributions, expanding the diversity of microbes that can perform denitrification. Phylogenetic analyses of the HCO superfamily demonstrate that nitric oxide reductases evolved multiple times independently from oxygen reductases, suggesting that complete denitrification evolved after aerobic respiration.

Two independent structures of the proton-pumping, respiratory cytochrome bo3 ubiquinol oxidase (cyt bo3) have been determined by cryogenic electron microscopy (cryo-EM) in styrene–maleic acid (SMA) copolymer nanodiscs and in membrane scaffold protein (MSP) nanodiscs to 2.55- and 2.19-A resolution, respectively. The structures include the metal redox centers (heme b, heme o3, and CuB), the redox-active cross-linked histidine–tyrosine cofactor, and the internal water molecules in the proton-conducting D channel. Each structure also contains one equivalent of ubiquinone-8 (UQ8) in the substrate binding site as well as several phospholipid molecules. The isoprene side chain of UQ8 is clamped within a hydrophobic groove in subunit I by transmembrane helix TM0, which is only present in quinol oxidases and not in the closely related cytochrome c oxidases. Both structures show carbonyl O1 of the UQ8 headgroup hydrogen bonded to D75I and R71I. In both structures, residue H98I occupies two conformations. In conformation 1, H98I forms a hydrogen bond with carbonyl O4 of the UQ8 headgroup, but in conformation 2, the imidazole side chain of H98I has flipped to form a hydrogen bond with E14I at the N-terminal end of TM0. We propose that H98I dynamics facilitate proton transfer from ubiquinol to the periplasmic aqueous phase during oxidation of the substrate. Computational studies show that TM0 creates a channel, allowing access of water to the ubiquinol headgroup and to H98I.

Cytochrome bo3, one of three terminal oxygen reductases in the aerobic respiratory chain of Escherichia coli, has been well characterized as a ubiquinol oxidase. The ability of cytochrome bo3 to catalyze the two-electron oxidation of ubiquinol-8 requires the enzyme to stabilize the one-electron oxidized ubisemiquinone species that is a transient intermediate in the reaction. Cytochrome bo3 has been shown recently to also utilize demethylmenaquinol-8 as a substrate that, along with menaquinol-8, replaces ubiquinol-8 when E. coli is grown under microaerobic or anaerobic conditions. In this work, we show that its steady-state turnover with 2,3-dimethyl-1,4-naphthoquinol, a water-soluble menaquinol analogue, is just as efficient as with ubiquinol-1. Using pulsed electron paramagnetic resonance spectroscopy, we demonstrate that the same residues in cytochrome bo3 that stabilize the semiquinone state of ubiquinone also stabilize the semiquinone state of menaquinone, with the hydrogen bond strengths and the dist...

Styrene-maleic acid (SMA) copolymers can extract membrane proteins from native membranes along with lipids as nanodiscs. Preparation with SMA is fast, cost-effective, and captures the native protein-lipid interactions. On the other hand, cryo-EM has become increasingly successful and efficient for structural determinations of membrane proteins, with biochemical sample preparation often the bottleneck. Three recent cryo-EM studies on the efflux transporter AcrB and the alternative complex III: cyt c oxidase supercomplex have demonstrated the potential of SMA nanodisc samples to yield high-resolution structure information of membrane proteins.

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify three families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA’ cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA’ from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA’ is the first isoform of cytbd containing only b-type hemes shown to be active when isolated from membranes, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

Cytochrome ba

Cytochrome bd-type oxygen reductases (cytbd) belong to one of three enzyme superfamilies that catalyze oxygen reduction to water. They are widely distributed in Bacteria and Archaea, but the full extent of their biochemical diversity is unknown. Here we used phylogenomics to identify 3 families and several subfamilies within the cytbd superfamily. The core architecture shared by all members of the superfamily consists of four transmembrane helices that bind two active site hemes, which are responsible for oxygen reduction. While previously characterized cytochrome bd-type oxygen reductases use quinol as an electron donor to reduce oxygen, sequence analysis shows that only one of the identified families has a conserved quinol binding site. The other families are missing this feature, suggesting that they use an alternative electron donor. Multiple gene duplication events were identified within the superfamily, resulting in significant evolutionary and structural diversity. The CydAA’ cytbd, found exclusively in Archaea, is formed by the co-association of two superfamily paralogs. We heterologously expressed CydAA’ from Caldivirga maquilingensis and demonstrated that it performs oxygen reduction with quinol as an electron donor. Strikingly, CydAA’ is the first isoform of cytbd containing only b-type hemes shown to be active when isolated, demonstrating that oxygen reductase activity in this superfamily is not dependent on heme d.

Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH2) oxidation, presumably fulfilling the role of cytochromes bc1/b6f in organisms that lack these enzymes. The molecular mechanism of ACIII is unknown and so far the complex has remained inaccessible for genetic modifications. The recently solved cryo-electron microscopy (cryo-EM) structures of ACIII from Flavobacterium johnsoniae, Rhodothermus marinus, and Roseiflexus castenholzii revealed no structural similarity to cytochrome bc1/b6f and there were variations in the heme-containing subunits ActA and ActE. These data implicated intriguing alternative electron transfer paths connecting ACIII with its redox partner, and left the contributions of ActE and the terminal domain of ActA to the catalytic mechanism unclear. Here, we report genetic deletion and complementation of F. johnsoniae actA and actE and the functional implications of such modifications. Deletion of actA led to the loss of activity of cytochrome aa3 (a redox partner of ACIII in this bacterium), which confirmed that ACIII is the sole source of electrons for this complex. Deletion of actE did not impair the activity of cytochrome aa3, revealing that ActE is not required for electron transfer between ACIII and cytochrome aa3. Nevertheless, absence of ActE negatively impacted the cell growth rate, pointing toward another, yet unidentified, function of this subunit. Possible explanations for these observations, including a proposal of a split in electron paths at the ActA/ActE interface, are discussed. The described system for genetic manipulations in F. johnsoniae ACIII offers new tools for studying the molecular mechanism of operation of this enzyme. IMPORTANCE Energy conversion is a fundamental process of all organisms, realized by specialized protein complexes, one of which is alternative complex III (ACIII). ACIII is a functional analogue of well-known mitochondrial complex III, but operates according to a different, still unknown mechanism. To understand how ACIII interacts functionally with its protein partners, we developed a genetic system to mutate the Flavobacterium johnsoniae genes encoding ACIII subunits. Deletion and complementation of heme-containing subunits revealed that ACIII is the sole source of electrons for cytochrome aa3 and that one of the redox-active subunits (ActE) is dispensable for electron transfer between these complexes. This study sheds light on the operation of the supercomplex of ACIII and cytochrome aa3 and suggests a division in the electron path within ACIII. It also shows a way to manipulate protein expression levels for application in other members of the Bacteroidetes phylum.

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc

A set of C43(DE3) and BL21(DE3) Escherichia coli host strains that are auxotrophic for various amino acids is briefly reviewed. These strains require the addition of a defined set of one or more amino acids in the growth medium, and have been specifically designed for overproduction of membrane or water-soluble proteins selectively labelled with stable isotopes, such as 2H, 13C and 15N. The strains described here are available for use and have been deposited into public strain banks. Although they cannot fully eliminate the possibility of isotope dilution and mixing, metabolic scrambling of the different amino acid types can be minimized through a careful consideration of the bacterial metabolic pathways. The use of a suitable auxotrophic expression host strain with an appropriately isotopically labelled growth medium ensures high levels of isotope labelling efficiency as well as selectivity for providing deeper insight into protein structure–function relationships.

The success of Staphylococcus aureus as a pathogen is due to its capability of fine‐tuning its cellular physiology to meet the challenges presented by diverse environments, which allows it to colonize multiple niches within a single vertebrate host. Elucidating the roles of energy‐yielding metabolic pathways could uncover attractive therapeutic strategies and targets. In this work, we seek to determine the effects of disabling NADH‐dependent aerobic respiration on the physiology of S. aureus. Differing from many pathogens, S. aureus has two type‐2 respiratory NADH dehydrogenases (NDH‐2s) but lacks the respiratory ion‐pumping NDHs. Here, we show that the NDH‐2s, individually or together, are not essential either for respiration or growth. Nevertheless, their absence eliminates biofilm formation, production of α‐toxin, and reduces the ability to colonize specific organs in a mouse model of systemic infection. Moreover, we demonstrate that the reason behind these phenotypes is the alteration of the fatty acid metabolism. Importantly, the SaeRS two‐component system, which responds to fatty acids regulation, is responsible for the link between NADH‐dependent respiration and virulence in S. aureus.

The heme‑copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O2 reduction and for proton pumping. This pathway, called the KC-pathway, starts at Glu-49P in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the KC-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pKa of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.

The coupling of the reaction of a tightly bound ubiquinone with the reduction of O2 in cytochrome bo3 of Escherichia coli was investigated. In the absence of the quinone, a strongly diminished rate of electrocatalytic reduction of oxygen is detected, which can be restored by adding quinones. The correlation of previous EPR data with the electrocatalytic study on mutations in the binding site at positions, Q101, D75, F93, H98, I102 and R71 reveal that the stabilization of the radical is not necessary for the oxygen reaction. The Q101 and F93 variants exhibit both well-defined catalytic i-V curves, whereas D75H, H98F, I102W and R71H exhibit broad i-V curves with large hysteresis pointing toward a strong alteration in their catalytic activity.

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria1–3. Like complex III (also known as the bc1 complex), ACIII catalyses the oxidation of membrane-bound quinol and the reduction of cytochrome c or an equivalent electron carrier. However, the two complexes have no structural similarity4–7. Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron–sulfur molybdoenzyme (CISM) superfamily8, including the proton pump polysulfide reductase9,10. We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer11–14, both as an independent enzyme and as a functional 1:1 supercomplex with an aa3-type cytochrome c oxidase (cyt aa3). We determined the structure of ACIII to 3.4 A resolution by cryo-electron microscopy and constructed an atomic model for its six subunits. The structure, which contains a [3Fe–4S] cluster, a [4Fe–4S] cluster and six haem c units, shows that ACIII uses known elements from other electron transport complexes arranged in a previously unknown manner. Modelling of the cyt aa3 component of the supercomplex revealed that it is structurally modified to facilitate association with ACIII, illustrating the importance of the supercomplex in this electron transport chain. The structure also resolves two of the subunits of ACIII that are anchored to the lipid bilayer with N-terminal triacylated cysteine residues, an important post-translational modification found in numerous prokaryotic membrane proteins that has not previously been observed structurally in a lipid bilayer. The structure of alternative complex III, a key enzyme in the bacterial electron transport chain, is reported both alone and as a supercomplex with an aa3-type cytochrome c oxidase.

Cytochrome aa3 is the terminal respiratory enzyme of all eukaryotes and many bacteria and archaea, reducing O2 to water and harnessing the free energy from the reaction to generate the transmembrane electrochemical potential. The diffusion of O2 to the heme-copper catalytic site, which is buried deep inside the enzyme, is the initiation step of the reaction chemistry. Our previous molecular dynamics (MD) study with cytochrome ba3, a homologous enzyme of cytochrome aa3 in Thermus thermophilus, demonstrated that O2 diffuses from the lipid bilayer to its reduction site through a 25 A long tunnel inferred by Xe binding sites detected by X-ray crystallography [Mahinthichaichan, P., Gennis, R., and Tajkhorshid, E. (2016) Biochemistry 55, 1265–1278]. Although a similar tunnel is observed in cytochrome aa3, this putative pathway appears partially occluded between the entrances and the reduction site. Also, the experimentally determined second-order rate constant for O2 delivery in cytochrome aa3 (∼108 M–1 s–1) is...

Respiration is carried out by a series of membrane-bound complexes in the inner mitochondrial membrane or in the cytoplasmic membrane of bacteria. Increasing evidence shows that these complexes organize into larger supercomplexes. In this work, we identified a supercomplex composed of cytochrome (cyt.) bc1 and aa3-type cyt. c oxidase in Rhodobacter sphaeroides. We purified the supercomplex using a His-tag on either of these complexes. The results from activity assays, native and denaturing PAGE, size exclusion chromatography, electron microscopy, optical absorption spectroscopy and kinetic studies on the purified samples support the formation and coupled quinol oxidation:O2 reduction activity of the cyt. bc1-aa3 supercomplex. The potential role of the membrane-anchored cyt. cy as a component in supercomplexes was also investigated.

Iron–sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4–n (n = 0, 1, 2) cluster by selective cysteine-13Cβ isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution...

The cytochrome bo3 ubiquinol oxidase is one of three respiratory oxygen reductases in the aerobic respiratory chain of Escherichia coli. The generally accepted model of catalysis assumes that cyt bo3 contains two distinct ubiquinol binding sites: (i) a low affinity (QL) site which is the traditional substrate binding site; and (ii) a high affinity (QH) site where a “permanently” bound quinone acts as a cofactor, taking two electrons from the substrate quinol and passing them one-by-one to the heme b component of the enzyme which, in turn, transfers them to the heme o3/CuB active site. Whereas the residues at the QH site are well defined, the location of the QL site remains unknown. The published X-ray structure does not contain quinone, and substantial amounts of the protein are missing as well. A recent bioinformatics study by Bossis et al. [Biochem J. (2014) 461, 305–314] identified a sequence motif G163EFX3GWX2Y173 as the likely QL site in the family of related quinol oxidases. In the current work, this was tested by site-directed mutagenesis. The results show that these residues are not important for catalytic function and do not define the QL substrate binding site.

The ba(3) cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic ...

Virtually all proton-pumping terminal respiratory oxygen reductases are members of the heme-copper oxidoreductase superfamily. Most of these enzymes use reduced cytochrome c as a source of electrons, but a group of enzymes have evolved to directly oxidize membrane-bound quinols, usually menaquinol or ubiquinol. All of the quinol oxidases have an additional transmembrane helix (TM0) in subunit I that is not present in the related cytochrome c oxidases. The current work reports the 3.6-Å-resolution X-ray structure of the cytochrome aa 3 -600 menaquinol oxidase from Bacillus subtilis containing 1 equivalent of menaquinone. The structure shows that TM0 forms part of a cleft to accommodate the menaquinol-7 substrate. Crystals which have been soaked with the quinol-analog inhibitor HQNO ( N -oxo-2-heptyl-4-hydroxyquinoline) or 3-iodo-HQNO reveal a single binding site where the inhibitor forms hydrogen bonds to amino acid residues shown previously by spectroscopic methods to interact with the semiquinone state of menaquinone, a catalytic intermediate.

Cytochrome

Hemes switch spots in a terminal oxidase Reduction of molecular oxygen to water is the driving force for respiration in aerobic organisms and is catalyzed by several distinct integral membrane complexes. These include an exclusively prokaryotic enzyme, cytochrome bd–type quinol oxidase, which is a potential antimicrobial target. Safarian et al. determined a high-resolution cryo–electron microscopy structure of this enzyme from the enteric bacterium Escherichia coli . Comparison to a homolog reveals a complete relocation of the site of oxygen binding and reduction caused by a change in the arrangement of heme cofactors and channels in the protein scaffold. This switch illustrates the diversity of structure and function in this family of enzymes and might reflect different biochemical roles of these homologs. Science , this issue p. 100

Sulfide:quinone oxidoreductase (SQR) is a monotopic membrane flavoprotein present in all domains of life, with multiple roles including sulfide detoxification, homeostasis and energy generation by providing electrons to respiratory or photosynthetic electron transport chains. A type III SQR from the hyperthermophilic archeon Caldivirga maquilingensis has been previously characterized, and its C-terminal amphipathic helices were demonstrated to be responsible for membrane binding. Here, the oligomeric state of this protein was experimentally evaluated by size exclusion chromatography, native gels and crosslinking, and found to be a monomer-dimer-trimer equilibrium. Remarkably, mutant and truncated variants unable to bind to the membrane are able to maintain their oligomeric association. Thus, unlike other related monotopic membrane proteins, the region involved in membrane binding does not influence oligomerization. Furthermore, by studying heterodimers between the WT and mutants, it was concluded that membrane binding requires an oligomer with at least two copies of the protein with intact C-terminal amphipathic helices. A structural homology model of the C. maquilingensis SQR was used to define the flavin- and quinone-binding sites. CmGly12, CmGly16, CmAla77 and CmPro44 were determined to be important for flavin binding. Unexpectedly, CmGly299 is only important for quinone reduction despite its proximity to bound FAD. CmPhe337 and CmPhe362 are also important for quinone binding apparently by direct interaction with the quinone ring, whereas CmLys359, postulated to hydrogen bond to the quinone, seems to have a more structural role. The results presented differentiate the Type III CmSQR from some of its counterparts classified as Type I, II and III.

PMID: 27664317; Cytochrome cbb3 (also known as C-type) oxidases belong to the family of heme-copper terminal oxidases which couple at the end of the respiratory chain the reduction of molecular oxygen into water and the pumping of protons across the membrane. They are expressed most often at low pressure of O2 and they exhibit a low homology of sequence with the cytochrome aa3 (A-type) oxidases found in mitochondria. Their binuclear active site comprises a high-spin heme b3 associated with a CuB center. The protein also contains one low-spin heme b and 3 hemes c. We address here the redox properties of cbb3 oxidases from three organisms, Rhodobacter sphaeroides, Vibrio cholerae and Pseudomonas stutzeri by means of electrochemical and spectroscopic techniques. We show that the redox potential of the heme b3 exhibits a relatively low midpoint potential, as in related cytochrome c-dependent nitric oxide reductases. Potential implications for the coupled electron transfer and proton uptake mechanism of C-type oxidases are discussed.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron–nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz′ = 11.8 MHz, whereas for H2, Tz′ = 8.6 MHz. Remarkabl...

Cytochrome bd, a component of the prokaryotic respiratory chain, is important under physiological stress and during pathogenicity. Electrons from quinol substrates are passed on via heme groups in the CydA subunit and used to reduce molecular oxygen. Close to the quinol binding site, CydA displays a periplasmic hydrophilic loop called Q-loop that is essential for quinol oxidation. In the carboxy-terminal part of this loop, CydA from Escherichia coli and other proteobacteria harbors an insert of ~60 residues with unknown function. In the current work, we demonstrate that growth of the multiple-deletion strain E. coli MB43∆cydA (∆cydA∆cydB∆appB∆cyoB∆nuoB) can be enhanced by transformation with E. coli cytochrome bd-I and we utilize this system for assessment of Q-loop mutants. Deletion of the cytochrome bd-I Q-loop insert abolished MB43∆cydA growth recovery. Swapping the cytochrome bd-I Q-loop for the Q-loop from Geobacillus thermodenitrificans or Mycobacterium tuberculosis CydA, which lack the insert, did not enhance the growth of MB43∆cydA, whereas swapping for the Q-loop from E. coli cytochrome bd-II recovered growth. Alanine scanning experiments identified the cytochrome bd-I Q-loop insert regions Ile318-Met322, Gln338-Asp342, Tyr353-Leu357, and Thr368-Ile372 as important for enzyme functionality. Those mutants that completely failed to recover growth of MB43∆cydA also lacked oxygen consumption activity and heme absorption peaks. Moreover, we were not able to isolate cytochrome bd-I from these inactive mutants. The results indicate that the cytochrome bd Q-loop exhibits low plasticity and that the Q-loop insert in E. coli is needed for complete, stable, assembly of cytochrome bd-I.

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn’s first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development., IMPORTANCE S. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target.

The coupling of the reaction of a tightly bound ubiquinone with the reduction of O

Microcin J25 (MccJ25), an antimicrobial peptide, targets the respiratory chain but the exact mechanism by which it does so remains unclear. Here, we reveal that MccJ25 is able to inhibit the enzymatic activity of the isolated cytochrome bd-I from E. coli and induces at the same time production of reactive oxygen species. MccJ25 behaves as a dose-dependent weak inhibitor. Intriguingly, MccJ25 is capable of producing a change in the oxidation state of cytochrome bd-I causing its partial reduction in the presence of cyanide. These effects are specific for cytochrome bd-I, since the peptide is not able to act on purified cytochrome bo3. Fil: Galván, Adriana Emilce. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Chalon, Miriam Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Ríos Colombo, Natalia Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina Fil: Schurig Briccio, Lici Ariane. University of Illinois; Estados Unidos Fil: Sosa Padilla Araujo, Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Gennis, Robert B.. University of Illinois; Estados Unidos Fil: Bellomio, Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Instituto Superior de Investigaciones Biológicas. Universidad Nacional de Tucumán. Instituto Superior de Investigaciones Biológicas; Argentina

The superfamily of heme-copper oxidoreductases (HCOs) include both NO and O(2) reductases. Nitric oxide reductases (NORs) are bacterial membrane enzymes that catalyze an intermediate step of denitrification by reducing nitric oxide (NO) to nitrous oxide (N(2)O). They are structurally similar to heme-copper oxygen reductases (HCOs), which reduce O(2) to water. The experimentally observed apparent bimolecular rate constant of NO delivery to the deeply buried catalytic site of NORs was previously reported to approach the diffusion-controlled limit (10(8)–10(9) M(−1)s(−1)). Using the crystal structure of cytochrome-c dependent NOR (cNOR) from Pseudomonas aeruginosa, we employed several protocols of molecular dynamics (MD) simulation, which include flooding simulations of NO molecules, implicit ligand sampling and umbrella sampling simulations, to elucidate how NO in solution accesses the catalytic site of this cNOR. The results show that NO partitions into the membrane, enters the enzyme from the lipid bilayer and diffuses to the catalytic site via a hydrophobic tunnel that is resolved in the crystal structures. This is similar to what has been found for O(2) diffusion through the closely related O(2) reductases. The apparent second order rate constant approximated using the simulation data is ~5×10(8) M(−1)s(−1), which is optimized by the dynamics of the amino acid side chains lining in the tunnel. It is concluded that both NO and O(2) reductases utilize well defined hydrophobic tunnels to assure that substrate diffusion to the buried catalytic sites is not rate limiting under physiological conditions.

Cytochrome aa(3) is the terminal respiratory enzyme of all eukaryotes and many bacteria and archaea, reducing O(2) to water and harnessing the free energy from the reaction to generate the transmembrane electrochemical potential. The diffusion of O(2) to the heme-copper catalytic site, which is buried deep inside the enzyme, is the initiation step of the reaction chemistry. Our previous molecular dynamics (MD) study with cytochrome ba(3), a homologous enzyme of cytochrome aa(3) in Thermus thermophilus, demonstrated that O(2) diffuses from the lipid bilayer to its reduction site through a 25-Å long tunnel inferred by Xe-binding sites detected by X-ray crystallography.(1) Although a similar tunnel is observed in cytochrome aa(3), this putative pathway appears partially occluded between the entrances and the reduction site. Also, the experimentally determined second-order rate constant for O(2) delivery in cytochrome aa(3) (~10(8) M(−1)s(−1)) is 10 times slower than that in cytochrome ba(3) (~10(9) M(−1)s(−1)). A question to be addressed is whether cytochrome aa(3) utilizes this X-ray inferred tunnel as the primary pathway for O(2) delivery. Using complimentary computational methods including multiple independent flooding MD simulations and implicit ligand sampling calculations, we probe the O(2) delivery pathways in cytochrome aa(3) of Rhodobacter sphaeroides. All of the O(2) molecules that arrived in the reduction site during the simulations were found to diffuse through the X-ray observed tunnel, despite its apparent constriction, supporting its role as the main O(2) delivery pathway in cytochrome aa(3). The rate constant for O(2) delivery in cytochrome aa(3), approximated using the simulation results, is 10 times slower than in cytochrome ba(3), in agreement with the experimentally determined rate constants.

The heme-copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O(2) reduction and for proton pumping. This pathway, called the K(C)-pathway, starts at Glu-49(P) in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the K(C)-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pK(a) of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B- family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.

Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating the structure-function relationship. Microseeding, the process of transferring sub-microscopic crystal nuclei from initial screens into new crystallization experiments, is an effective, yet underutilized approach to grow crystals suitable for X-ray crystallography. Here, we report simplified methods for crystallization of membrane proteins that utilize microseeding in X ray transparent microfluidic chips. First, a microfluidic method for introduction of microseed dilutions into metastable crystallization experiments is demonstrated for photoactive yellow protein and cytochrome bo(3) oxidase. As microseed concentration decreased, the number of crystals decreased while the average size increased. Second, we demonstarte a microfluidic chip for microseed screening, where many crystallization conditions were formulated on-chip prior to mixing with microseeds. Crystallization composition, crystal size, and diffraction data were collected and mapped on phase diagrams, which revelaed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.

The recent X-ray structure of the cytochrome bd respiratory oxygen reductase showed that two of the three heme components, heme d and heme b(595), have glutamic acid as an axial ligand. No other native heme proteins are known to have glutamic acid axial ligands. In this work, site-directed mutagenesis is used to probe the roles of these glutamic acids, E445 and E99 in the E. coli enzyme. It is concluded that neither glutamate is a strong ligand to the heme Fe and they are not the major determinates of heme binding to the protein. Although very important, neither glutamate is absolutely essential for catalytic function. The close interactions between the three hemes in cyt bd result in highly cooperative properties. For example, mutation of E445, which is near heme d, has its greatest effects on the properties of heme b(595) and heme b(558). It is concluded that 1) O(2) binds to the hydrophilic side of heme d and displaces E445; 2) E445 forms a salt bridge with R448 within the O(2) binding pocket, and both residues play a role to stabilize oxygenated states of heme d during catalysis; 3) E445 and E99 are each protonated accompanying electron transfer to heme d and heme b(595), respectively; 4) All protons used to generate water within the heme d active site come from the cytoplasm and are delivered through a channel that must include internal water molecules to assist proton transfer: [cytoplasm]→E107→E99 (heme b(595))→E445 (heme d)→oxygenated heme d.

The C-family (cbb3) of heme-copper oxygen reductases are proton-pumping enzymes terminating the aerobic respiratory chains of many bacteria, including a number of human pathogens. The most common form of these enzymes contains one copy each of 4 subunits encoded by the ccoNOQP operon. In the cbb3 from Rhodobacter capsulatus, the enzyme is assembled in a stepwise manner, with an essential role played by an assembly protein CcoH. Importantly, it has been proposed that a transient interaction between the transmembrane domains of CcoP and CcoH is essential for assembly. Here, we test this proposal by showing that a genetically engineered form of cbb3 from Vibrio cholerae (CcoNOQPX) that lacks the hydrophilic domain of CcoP, where the two heme c moieties are present, is fully assembled and stable. Single-turnover kinetics of the reaction between the fully reduced CcoNOQPX and O2 are essentially the same as the wild type enzyme in oxidizing the 4 remaining redox-active sites. The enzyme retains approximately 10% of the steady state oxidase activity using the artificial electron donor TMPD, but has no activity using the physiological electron donor cytochrome c4, since the docking site for this cytochrome is presumably located on the absent domain of CcoP. Residue E49 in the hydrophobic domain of CcoP is the entrance of the KC-channel for proton input, and the E49A mutation in the truncated enzyme further reduces the steady state activity to less than 3%. Hence, the same proton channel is used by both the wild type and truncated enzymes.

Cytochrome aa3-600 is a terminal oxidase in the electron transport pathway that contributes to the electrochemical membrane potential by actively pumping protons. A notable feature of this enzyme complex is that it uses menaquinol as its electron donor instead of cytochrome c when it reduces dioxygen to water. The enzyme stabilizes a menasemiquinone radical (SQ) at a high affinity site that is important for catalysis. One of the residues that interacts with the semiquinone is Arg70. We have made the R70H mutant and have characterized the menasemiquinone radical by advanced X- and Q-band EPR. The bound SQ of the R70H mutant exhibits a strong isotropic hyperfine coupling (a(14)N ≈ 2.0 MHz) with a hydrogen bonded nitrogen. This nitrogen originates from a histidine side chain, based on its quadrupole coupling constant, e(2)qQ/h = 1.44 MHz, typical for protonated imidazole nitrogens. In the wild-type cyt aa3-600, the SQ is instead hydrogen bonded with Nε from the Arg70 side chain. Analysis of the (1)H 2D electron spin echo envelope modulation (ESEEM) spectra shows that the mutation also changes the number and strength of the hydrogen bonds between the SQ and the surrounding protein. Despite the alterations in the immediate environment of the SQ, the R70H mutant remains catalytically active. These findings are in contrast to the equivalent mutation in the close homologue, cytochrome bo3 ubiquinol oxidase from Escherichia coli, where the R71H mutation eliminates function.

The membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP+ by NADH to proton translocation. Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al., 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP+/NADPH during enzyme turnover.