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Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration., Competing Interests: Competing interests statement:The authors declare no competing interest.

Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (© 2021. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Understanding the mechanistic coupling of molecular oxygen reduction and proton pumping for adenosine triphosphate synthesis during cellular respiration is the primary goal of research on heme-copper oxidases—the terminal complex in the membrane-bound electron transport chain. Cleavage of the oxygen-oxygen bond by the heme-copper oxidases forms the key intermediate P M , which initiates proton pumping. This intermediate is now experimentally defined by variable-temperature, variable-field magnetic circular dichroism spectroscopy on a previously unobserved excited state feature associated with its heme iron(IV)-oxo center. These data provide evidence that the iron(IV)-oxo in P M is magnetically coupled to both a copper(II) and a cross-linked tyrosyl radical in the active site. These results provide new insight into the oxygen-oxygen bond cleavage and proton-pumping mechanisms of heme-copper oxidases.

Bacterial alternative complex III (ACIII) catalyzes menaquinol (MKH 2 ) oxidation, presumably fulfilling the role of cytochromes bc 1 / b 6 f 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 bc 1 / b 6 f 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 aa 3 (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 aa 3 , revealing that ActE is not required for electron transfer between ACIII and cytochrome aa 3 . 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 aa 3 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 aa 3 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.

Cytochrome ba 3 from Thermus thermophilus belongs to the B family of heme-copper oxidases and pumps protons across the membrane with an as yet unknown mechanism. The K channel of the A family heme-copper oxidases provides delivery of a substrate proton from the internal water phase to the binuclear heme-copper center (BNC) during the reductive phase of the catalytic cycle, while the D channel is responsible for transferring both substrate and pumped protons. By contrast, in the B family oxidases there is no D-channel and the structural equivalent of the K channel seems to be responsible for the transfer of both categories of protons. Here we have studied the effect of the T315V substitution in the K channel on the kinetics of membrane potential generation coupled to the oxidative half-reaction of the catalytic cycle of cytochrome ba 3 . The results suggest that the mutated enzyme does not pump protons during the reaction of the fully reduced form with molecular oxygen in a single turnover. Specific inhibition of proton pumping in the T315V mutant appears to be a consequence of inability to provide rapid (τ ~ 100 μs) reprotonation of the internal transient proton donor(s) of the K channel. In contrast to the A family, the K channel of the B-type oxidases is necessary for the electrogenic transfer of both pumped and substrate protons during the oxidative half-reaction of the catalytic cycle., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 1 and aa 3 -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:O 2 reduction activity of the cyt. bc 1 -aa 3 supercomplex. The potential role of the membrane-anchored cyt. c y as a component in supercomplexes was also investigated., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

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 Author(s) 2020. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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 IC 50 s 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 IC 50 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.

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 Ile 318 -Met 322 , Gln 338 -Asp 342 , Tyr 353 -Leu 357 , and Thr 368 -Ile 372 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

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., (© 2020 The Authors.)

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 V., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no competing interest.

Cytochrome bo 3 , 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 bo 3 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 bo 3 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 bo 3 that stabilize the semiquinone state of ubiquinone also stabilize the semiquinone state of menaquinone, with the hydrogen bond strengths and the distribution of unpaired spin density accommodated for the different substrate. Catalytic function with menaquinol is more tolerant of mutations at the active site than with ubiquinol. A mutation of one of the stabilizing residues (R71H in subunit I) that eliminates the ubiquinol oxidase activity of cytochrome bo 3 does not abolish activity with soluble menaquinol analogues.

The crystal structure of the enzyme previously characterized as a type-2 NADH:menaquinone oxidoreductase (NDH-2) from Thermus thermophilus has been solved at a resolution of 2.9 Å and revealed that this protein is, in fact, a coenzyme A-disulfide reductase (CoADR). Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Thermus thermophilus and is maintained in the reduced state by this enzyme (CoADR). Although the enzyme does exhibit NADH:menadione oxidoreductase activity expected for NDH-2 enzymes, the specific activity with CoAD as an electron acceptor is about 5-fold higher than with menadione. Furthermore, the crystal structure contains coenzyme A covalently linked Cys44, a catalytic intermediate (Cys44-S-S-CoA) reduced by NADH via the FAD cofactor. Soaking the crystals with menadione shows that menadione can bind to a site near the redox active FAD, consistent with the observed NADH:menadione oxidoreductase activity. CoADRs from other species were also examined and shown to have measurable NADH:menadione oxidoreductase activity. Although a common feature of this family of enzymes, no biological relevance is proposed. The CoADR from T. thermophilus is a soluble homodimeric enzyme. Expression of the recombinant TtCoADR at high levels in E. coli results in a small fraction that co-purifies with the membrane fraction, which was used previously to isolate the enzyme wrongly identified as a membrane-bound NDH-2. It is concluded that T. thermophilus does not contain an authentic NDH-2 component in its aerobic respiratory chain., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cytochrome bd-type quinol oxidases catalyze the reduction of molecular oxygen to water in the respiratory chain of many human-pathogenic bacteria. They are structurally unrelated to mitochondrial cytochrome c oxidases and are therefore a prime target for the development of antimicrobial drugs. We determined the structure of the Escherichia coli cytochrome bd-I oxidase by single-particle cryo-electron microscopy to a resolution of 2.7 angstroms. Our structure contains a previously unknown accessory subunit CydH, the L-subfamily-specific Q-loop domain, a structural ubiquinone-8 cofactor, an active-site density interpreted as dioxygen, distinct water-filled proton channels, and an oxygen-conducting pathway. Comparison with another cytochrome bd oxidase reveals structural divergence in the family, including rearrangement of high-spin hemes and conformational adaption of a transmembrane helix to generate a distinct oxygen-binding site., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 bo 3 ., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The coupling of the reaction of a tightly bound ubiquinone with the reduction of O 2 in cytochrome bo 3 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., (© 2018 Federation of European Biochemical Societies.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Bedaquiline (BDQ), an inhibitor of the mycobacterial F 1 F o -ATP synthase, has revolutionized the antitubercular drug discovery program by defining energy metabolism as a potent new target space. Several studies have recently suggested that BDQ ultimately causes mycobacterial cell death through a phenomenon known as uncoupling. The biochemical basis underlying this, in BDQ, is unresolved and may represent a new pathway to the development of effective therapeutics. In this communication, we demonstrate that BDQ can inhibit ATP synthesis in Escherichia coli by functioning as a H + /K + ionophore, causing transmembrane pH and potassium gradients to be equilibrated. Despite the apparent lack of a BDQ-binding site, incorporating the E. coli F o subunit into liposomes enhanced the ionophoric activity of BDQ. We discuss the possibility that localization of BDQ at F 1 F o -ATP synthases enables BDQ to create an uncoupled microenvironment, by antiporting H + /K + Ionophoric properties may be desirable in high-affinity antimicrobials targeting integral membrane proteins., Competing Interests: The authors declare no conflict of interest.

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., (Copyright © 2018 Lencina et al.)

Cytochrome c oxidase catalyzes reduction of O 2 to H 2 O at a catalytic site that is composed of a copper ion and heme group. The reaction is linked to translocation of four protons across the membrane for each O 2 reduced to water. The free energy associated with electron transfer to the catalytic site is unequal for the four electron-transfer events. Most notably, the free energy associated with reduction of the catalytic site in the oxidized cytochrome c oxidase (state O) is not sufficient for proton pumping across the energized membrane. Yet, this electron transfer is mechanistically linked to proton pumping. To resolve this apparent discrepancy, a high-energy oxidized state (denoted O H ) was postulated and suggested to be populated only during catalytic turnover. The difference between states O and O H was suggested to be manifested in an elevated midpoint potential of Cu B in the latter. This proposal predicts that one-electron reduction of cytochrome c oxidase after its oxidation would yield re-reduction of essentially only Cu B . Here, we investigated this process and found ~5% and ~6% reduction of heme a 3 and Cu B , respectively, i.e. the apparent redox potentials for heme a 3 and Cu B are lower than that of heme a.

Alternative complex III (ACIII) is a key component of the respiratory and/or photosynthetic electron transport chains of many bacteria 1-3 . Like complex III (also known as the bc 1 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 similarity 4-7 . Although ACIII has eluded structural characterization, several of its subunits are known to be homologous to members of the complex iron-sulfur molybdoenzyme (CISM) superfamily 8 , including the proton pump polysulfide reductase 9,10 . We isolated the ACIII from Flavobacterium johnsoniae with native lipids using styrene maleic acid copolymer 11-14 , both as an independent enzyme and as a functional 1:1 supercomplex with an aa 3 -type cytochrome c oxidase (cyt aa 3 ). We determined the structure of ACIII to 3.4 Å 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 aa 3 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.

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 [Mahinthichaichan, P., Gennis, R., and Tajkhorshid, E. (2016) Biochemistry 55, 1265-1278]. 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 complementary 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.

Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating structure-function relationships. 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 demonstrate 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 revealed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.

Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo 3 was analyzed. The mutant strains lacking cytochrome bo 3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo 3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo 3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25., (Copyright © 2017. Published by Elsevier B.V.)

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- 13 C β isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe 2+ ligands than the nonreducible Fe 3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe 2+ 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 of all cysteine- 13 C β hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

The nicotinamide nucleotide transhydrogenase (TH) is an integral membrane enzyme that uses the proton-motive force to drive hydride transfer from NADH to NADP + in bacteria and eukaryotes. Here we solved a 2.2-Å crystal structure of the TH transmembrane domain (Thermus thermophilus) at pH 6.5. This structure exhibits conformational changes of helix positions from a previous structure solved at pH 8.5, and reveals internal water molecules interacting with residues implicated in proton translocation. Together with molecular dynamics simulations, we show that transient water flows across a narrow pore and a hydrophobic "dry" region in the middle of the membrane channel, with key residues His42 α2 (chain A) being protonated and Thr214 β (chain B) displaying a conformational change, respectively, to gate the channel access to both cytoplasmic and periplasmic chambers. Mutation of Thr214 β to Ala deactivated the enzyme. These data provide new insights into the gating mechanism of proton translocation in TH., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Cytochrome bo 3 is a respiratory proton-pumping oxygen reductase that is a member of the heme-copper superfamily that utilizes ubiquinol-8 (Q 8 H 2 ) as a substrate. The current consensus model has Q 8 H 2 oxidized at a low affinity site (Q L ), passing electrons to a tightly bound quinone cofactor at a high affinity site (Q H site) that stabilizes the one-electron reduced ubisemiquinone, facilitating the transfer of electrons to the redox active metal centers where O 2 is reduced to water. The current work shows that the Q 8 bound to the Q H site is more dynamic than previously thought. In addition, mutations of residues at the Q H site that do not abolish activity have been re-examined and shown to have properties expected of mutations at the substrate binding site (Q L ): an increase in the K M of the substrate ubiquinol-1 (up to 4-fold) and an increase in the apparent K i of the inhibitor HQNO (up to 8-fold). The data suggest that there is only one binding site for ubiquinol in cyt bo 3 and that site corresponds to the Q H site.

The cytochrome bo 3 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 bo 3 contains two distinct ubiquinol binding sites: (i) a low affinity (Q L ) site which is the traditional substrate binding site; and (ii) a high affinity (Q H ) 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 o 3 /Cu B active site. Whereas the residues at the Q H site are well defined, the location of the Q L 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 G 163 EFX 3 GWX 2 Y 173 as the likely Q L 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 Q L substrate binding site., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 Å.

Cytochrome cbb 3 (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 O 2 and they exhibit a low homology of sequence with the cytochrome aa 3 (A-type) oxidases found in mitochondria. Their binuclear active site comprises a high-spin heme b 3 associated with a Cu B center. The protein also contains one low-spin heme b and 3 hemes c. We address here the redox properties of cbb 3 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 b 3 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., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The respiratory cytochrome bo 3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQ H ), which is a transient intermediate during the electron-mediated reduction of O 2 to water. It is known that SQ H 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, SQ H was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1 H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo 3 in a H 2 O 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 T z' = 11.8 MHz, whereas for H2, T z' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo 3 Q H site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

Unlabelled: Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, osteomyelitis, and endocarditis in the developed world. The ability of S. aureus to cause substantial disease in distinct host environments is supported by a flexible metabolism that allows this pathogen to overcome challenges unique to each host organ. One feature of staphylococcal metabolic flexibility is a branched aerobic respiratory chain composed of multiple terminal oxidases. Whereas previous biochemical and spectroscopic studies reported the presence of three different respiratory oxygen reductases (o type, bd type, and aa3 type), the genome contains genes encoding only two respiratory oxygen reductases, cydAB and qoxABCD Previous investigation showed that cydAB and qoxABCD are required to colonize specific host organs, the murine heart and liver, respectively. This work seeks to clarify the relationship between the genetic studies showing the unique roles of the cydAB and qoxABCD in virulence and the respiratory reductases reported in the literature. We establish that QoxABCD is an aa3-type menaquinol oxidase but that this enzyme is promiscuous in that it can assemble as a bo3-type menaquinol oxidase. However, the bo3 form of QoxABCD restricts the carbon sources that can support the growth of S. aureus In addition, QoxABCD function is supported by a previously uncharacterized protein, which we have named CtaM, that is conserved in aerobically respiring Firmicutes In total, these studies establish the heme A biosynthesis pathway in S. aureus, determine that QoxABCD is a type aa3 menaquinol oxidase, and reveal CtaM as a new protein required for type aa3 menaquinol oxidase function in multiple bacterial genera., Importance: Staphylococcus aureus relies upon the function of two terminal oxidases, CydAB and QoxABCD, to aerobically respire and colonize distinct host tissues. Previous biochemical studies support the conclusion that a third terminal oxidase is also present. We establish the components of the S. aureus electron transport chain by determining the heme cofactors that interact with QoxABCD. This insight explains previous observations by revealing that QoxABCD can utilize different heme cofactors and confirms that the electron transport chain of S. aureus is comprised of two terminal menaquinol oxidases. In addition, a newly identified protein, CtaM, is found to be required for the function of QoxABCD. These results provide a more complete assessment of the molecular mechanisms that support staphylococcal respiration., (Copyright © 2016 Hammer et al.)

Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M(-1) cm(-1)) and 558 nm (2290 M(-1) cm(-1)) in the UV-vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10(-4) cm(-1), and another more rhombic, with a smaller A∥ value of 69 × 10(-4) cm(-1). The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV-vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.

In the ligand channel of the cytochrome c oxidase from Rhodobacter sphaeroides (Rs aa3 ) W172 and F282 have been proposed to generate a constriction that may slow ligand access to and from the active site. To explore this issue, the tryptophan and phenylalanine residues in Rs aa3 were mutated to the less bulky tyrosine and threonine residues, respectively, which occupy these sites in Thermus thermophilus (Tt) ba3 cytochrome oxidase. The CO photolysis and recombination dynamics of the reduced wild-type Rs aa3 and the W172Y/F282T mutant were investigated using time-resolved optical absorption spectroscopy. The spectral changes associated with the multiple processes are attributed to different conformers. The major CO recombination process (44 μs) in the W172Y/F282T mutant is ~500 times faster than the predominant CO recombination process in the wild-type enzyme (~23 ms). Classical dynamic simulations of the wild-type enzyme and double mutant showed significant structural changes at the active site in the mutant, including movement of the heme a3 ring-D propionate toward CuB and reduced binuclear center cavity volume. These structural changes effectively close the ligand exit pathway from the binuclear center, providing a basis for the faster CO recombination in the double mutant., (© 2016 The American Society of Photobiology.)