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Background NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. Results We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that “plugs” the NorQ ring pore to the VWA domain of NorD with a protruding “finger” inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples. Conclusions Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

Background Mitochondrial respiration is organized in a series of enzyme complexes in turn forming dynamic supercomplexes. In Saccharomyces cerevisiae (baker’s yeast), Cox13 (CoxVIa in mammals) is a conserved peripheral subunit of Complex IV (cytochrome c oxidase, CytcO), localized at the interface of dimeric bovine CytcO, which has been implicated in the regulation of the complex. Results Here, we report the solution NMR structure of Cox13, which forms a dimer in detergent micelles. Each Cox13 monomer has three short helices (SH), corresponding to disordered regions in X-ray or cryo-EM structures of homologous proteins. Dimer formation is mainly induced by hydrophobic interactions between the transmembrane (TM) helix of each monomer. Furthermore, an analysis of chemical shift changes upon addition of ATP revealed that ATP binds at a conserved region of the C terminus with considerable conformational flexibility. Conclusions Together with functional analysis of purified CytcO, we suggest that this ATP interaction is inhibitory of catalytic activity. Our results shed light on the structural flexibility of an important subunit of yeast CytcO and provide structure-based insight into how ATP could regulate mitochondrial respiration.

Intrinsic apoptosis as a modality of regulated cell death is intimately linked to permeabilization of the outer mitochondrial membrane and subsequent release of the protein cytochrome c into the cytosol, where it can participate in caspase activation via apoptosome formation. Interestingly, cytochrome c release is an ancient feature of regulated cell death even in unicellular eukaryotes that do not contain an apoptosome. Therefore, it was speculated that cytochrome c release might have an additional, more fundamental role for cell death signalling, because its absence from mitochondria disrupts oxidative phosphorylation. Here, we permanently anchored cytochrome c with a transmembrane segment to the inner mitochondrial membrane of the yeast Saccharomyces cerevisiae, thereby inhibiting its release from mitochondria during regulated cell death. This cytochrome c retains respiratory growth and correct assembly of mitochondrial respiratory chain supercomplexes. However, membrane anchoring leads to a sensitisation to acetic acid-induced cell death and increased oxidative stress, a compensatory elevation of cellular oxygen-consumption in aged cells and a decreased chronological lifespan. We therefore conclude that loss of cytochrome c from mitochondria during regulated cell death and the subsequent disruption of oxidative phosphorylation is not required for efficient execution of cell death in yeast, and that mobility of cytochrome c within the mitochondrial intermembrane space confers a fitness advantage that overcomes a potential role in regulated cell death signalling in the absence of an apoptosome.

A key step of denitrification, the reduction of toxic nitric oxide to nitrous oxide, is catalysed by cytochrome c-dependent NO reductase (cNOR). cNOR contains four redox-active cofactors: three hemes and a nonheme iron (FeB ). Heme b3 and FeB constitute the active site, but the specific mechanism of NO-binding events and reduction is under debate. Here, we used a recently constructed, fully folded and hemylated cNOR variant that lacks FeB to investigate the role of FeB during catalysis. We show that in the FeB -less cNOR, binding of both NO and O2 to heme b3 still occurs but further reduction is impaired, although to a lesser degree for O2 than for NO. Implications for the catalytic mechanisms of cNOR are discussed.

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.

Bacterial NO reductases (NOR) catalyze the reduction of NO into N2O, either as a step in denitrification or as a detoxification mechanism. cNOR from Paracoccus (P.) denitrificans is expressed from the norCBQDEF operon, but only the NorB and NorC proteins are found in the purified NOR complex. Here, we established a new purification method for the P. denitrificans cNOR via a His-tag using heterologous expression in E. coli. The His-tagged enzyme is both structurally and functionally very similar to non-tagged cNOR. We were also able to express and purify cNOR from the structural genes norCB only, in absence of the accessory genes norQDEF. The produced protein is a stable NorCB complex containing all hemes and it can bind gaseous ligands (CO) to heme b3, but it is catalytically inactive. We show that this deficient cNOR lacks the non-heme iron cofactor FeB. Mutational analysis of the nor gene cluster revealed that it is the norQ and norD genes that are essential to form functional cNOR. NorQ belongs to the family of MoxR P-loop AAA+ ATPases, which are in general considered to facilitate enzyme activation processes often involving metal insertion. Our data indicates that NorQ and NorD work together in order to facilitate non-heme Fe insertion. This is noteworthy since in many cases Fe cofactor binding occurs spontaneously. We further suggest a model for NorQ/D-facilitated metal insertion into cNOR.

Respiration in Saccharomyces cerevisiae is regulated by small proteins such as the respiratory supercomplex factors (Rcf). One of these factors (Rcf1) has been shown to interact with complexes III (cyt. bc1) and IV (cytochrome c oxidase, CytcO) of the respiratory chain and to modulate the activity of the latter. Here, we investigated the effect of deleting Rcf1 on the functionality of CytcO, purified using a protein C-tag on core subunit 1 (Cox1). Specifically, we measured the kinetics of ligand binding to the CytcO catalytic site, the O2-reduction activity and changes in light absorption spectra. We found that upon removal of Rcf1 a fraction of the CytcO is incorrectly assembled with structural changes at the catalytic site. The data indicate that Rcf1 modulates the assembly and activity of CytcO by shifting the equilibrium of structural sub-states toward the fully active, intact form.

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O2-binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O2, and found that the pKa of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed.

Cytochrome c oxidase is a membrane-bound redox-driven proton pump that harbors two proton-transfer pathways, D and K, which are used at different stages of the reaction cycle. Here, we address the question if a D pathway with a modified energy landscape for proton transfer could take over the role of the K pathway when the latter is blocked by a mutation. Our data indicate that structural alterations near the entrance of the D pathway modulate energy barriers that influence proton transfer to the proton-loading site. The data also suggest that during reduction of the catalytic site, its protonation has to occur via the K pathway and that this proton transfer to the catalytic site cannot take place through the D pathway.

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.

Kinetic methods used to investigate electron and proton transfer within cytochrome c oxidase (Cyt c O) are often based on the use of light to dissociate small ligands, such as CO, thereby initiating the reaction. Studies of intact mitochondria using these methods require identification of proteins that may bind CO and determination of the ligand-binding kinetics. In the present study we have investigated the kinetics of CO-ligand binding to S. cerevisiae mitochondria and cellular extracts. The data indicate that CO binds to two proteins, Cyt c O and a (yeast) flavohemoglobin (yHb). The latter has been shown previously to reside in both the cell cytosol and the mitochondrial matrix. Here, we found that yHb resides also in the intermembrane space and binds CO in its reduced state. As observed previously, we found that the yHb population in the mitochondrial matrix binds CO, but only after removal of the inner membrane. The mitochondrial yHb (in both the intermembrane space and the matrix) recombines with CO with τ ≅ 270 ms, which is significantly slower than observed with the cytosolic yHb (main component τ ≅ 1.3 ms). The data indicate that the yHb populations in the different cell compartments differ in structure.

Cytochrome c oxidases (CcO) reduce O2 to H2O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of CcO can also reduce NO to N2O and water while others cannot. Here, the mechanism for NO-reduction in CcO is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a "true" cytochrome c-dependent NO reductase (cNOR). The calculations show that in cNOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in CcO lead to a slower or even impossible reaction, consistent with experimental observations. In both enzymes the reaction is initiated by addition of two NO molecules to the reduced active site, forming a hyponitrite intermediate. In cNOR, N2O can then be formed using only the active-site electrons. In contrast, in CcO, one proton-coupled reduction step most likely has to occur before N2O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different CcO species with the same heme a3-Cu active site differ with respect to NO reduction efficiency, since they have a varying number and/or properties of proton channels. Finally, the calculations also indicate that a conserved active site valine plays a role in reducing the rate of NO reduction in CcO.

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.

The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

We used the amphipathic styrene maleic acid (SMA) co-polymer to extract cytochrome c oxidase (CytcO) in its native lipid environment from S. cerevisiae mitochondria. Native nanodiscs containing one CytcO per disc were purified using affinity chromatography. The longest cross-sections of the native nanodiscs were 11 nm × 14 nm. Based on this size we estimated that each CytcO was surrounded by ~100 phospholipids. The native nanodiscs contained the same major phospholipids as those found in the mitochondrial inner membrane. Even though CytcO forms a supercomplex with cytochrome bc(1) in the mitochondrial membrane, cyt. bc(1) was not found in the native nanodiscs. Yet, the loosely-bound Respiratory SuperComplex factors were found to associate with the isolated CytcO. The native nanodiscs displayed an O(2)-reduction activity of ~130 electrons CytcO(−1) s(−1) and the kinetics of the reaction of the fully reduced CytcO with O(2) was essentially the same as that observed with CytcO in mitochondrial membranes. The kinetics of CO-ligand binding to the CytcO catalytic site was similar in the native nanodiscs and the mitochondrial membranes. We also found that excess SMA reversibly inhibited the catalytic activity of the mitochondrial CytcO, presumably by interfering with cyt. c binding. These data point to the importance of removing excess SMA after extraction of the membrane protein. Taken together, our data shows the high potential of using SMA-extracted CytcO for functional and structural studies.

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.

As outlined by Peter Mitchell in the chemiosmotic theory, an intermediate in energy conversion in biological systems is a proton electrochemical potential difference (“proton gradient”) across a membrane, generated by membrane-bound protein complexes. These protein complexes accommodate proton-transfer pathways through which protons are conducted. In this review, we focus specifically on the role of the protein–membrane surface and the surface–bulk water interface in the dynamics of proton delivery to these proton-transfer pathways. The general mechanisms are illustrated by experimental results from studies of bacterial photosynthetic reaction centres (RCs) and cytochrome c oxidase (CcO).

C-type heme-copper oxidases terminate the respiratory chain in many pathogenic bacteria, and will encounter elevated concentrations of NO produced by the immune defense of the host. Thus, a decreased sensitivity to NO in C-type oxidases would increase the survival of these pathogens. Here we have compared the inhibitory effect of NO in C-type oxidases to that in the mitochondrial A-type. We show that O2-reduction in both the Rhodobacter sphaeroides and Vibrio cholerae C-type oxidases is strongly and reversibly inhibited by submicromolar NO, with an inhibition pattern similar to the A-type. Thus, NO tolerance in pathogens with a C-type terminal oxidase has to rely mainly on other mechanisms.

We have investigated the electron-proton coupling during the peroxy (P(R)) to oxo-ferryl (F) and F to oxidised (O) transitions in cytochrome c oxidase from Rhodobacter sphaeroides. The kinetics of these reactions were investigated in two different mutant enzymes: (1) ED(I-286), in which one of the key residues in the D-pathway, E(I-286), was replaced by an aspartate which has a shorter side chain than that of the glutamate and, (2) ML(II-263), in which the redox potential of Cu(A) is increased by approximately 100 mV, which slows electron transfer to the binuclear centre during the F-->O transition by a factor of approximately 200. In ED(I-286) proton uptake during P(R)-->F was slowed by a factor of approximately 5, which indicates that E(I-286) is the proton donor to P(R). In addition, in the mutant enzyme the F-->O transition rate displayed a deuterium isotope effect of approximately 2.5 as compared with approximately 7 in the wild-type enzyme. Since the entire deuterium isotope effect was shown to be associated with a single proton-transfer reaction in which the proton donor and acceptor must approach each other (M. Karpefors, P. Adelroth, P. Brzezinski, Biochemistry 39 (2000) 6850), the smaller deuterium isotope effect in ED(I-286) indicates that proton transfer from E(I-286) determines the rate also of the F-->O transition. In ML(II-263) the electron-transfer to the binuclear centre is slower than the intrinsic proton-transfer rate through the D-pathway. Nevertheless, both electron and proton transfer to the binuclear centre displayed a deuterium isotope effect of approximately 8, i.e., about the same as in the wild-type enzyme, which shows that these reactions are intimately coupled.

During the last few years our knowledge of the structure and function of heme copper oxidases has greatly profited from the use of site-directed mutagenesis in combination with biophysical techniques. This, together with the recently-determined crystal structures of cytochrome c oxidase, has now made it possible to design experiments aimed at targeting specific pump mechanisms. Here, we summarize results from our recent kinetic studies of electron and proton-transfer reactions in wild-type and mutant forms of cytochrome c oxidase from Rhodobacter sphaeroides. These studies have made it possible to identify amino acid residues involved in proton transfer during specific reaction steps and provide a basis for discussion of mechanisms of electron and proton transfer in terminal oxidases. The results indicate that the pathway through K(I-362)/T(I-359), but not through D(I-132)/E(I-286), is used for proton transfer to a protonatable group interacting electrostatically with heme a3, i.e., upon reduction of the binuclear center. The pathway through D(I-132)/E(I-286) is used for uptake of pumped and substrate protons during the pumping steps during O2 reduction.

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246]. In this study, we have investigated the reaction between this qNOR and oxygen. Our results show that, like some cNORs, the G. stearothermophilus qNOR is capable of O2 reduction with a turnover of ~3electronss−1 at 40°C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) qNOR reacts with oxygen in a reaction with a time constant of 1.8ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between qNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in qNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Heme–copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases.

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N(2)O) (2NO + 2H(+) + 2e(-) -->N(2)O + H(2)O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b(3), and a non-heme iron Fe(B). Here, we have studied the reaction between fully reduced NOR and NO using the "flow-flash" technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b(3) occurs with a time constant of approximately 1 micros at 1.5 mM NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe(B) before ligation to heme b(3). Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s(-1) and 3 s(-1) at 1.5 mM NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s(-1) and 12 s(-1) at 75 microM NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.

The bacterial respiratory nitric-oxide reductase (NOR) is a member of the superfamily of O(2)-reducing, proton-pumping, heme-copper oxidases. Even although nitric oxide reduction is a highly exergonic reaction, NOR is not a proton pump and rather than taking up protons from the cytoplasmic (membrane potential-negative) side of the membrane, like the heme-copper oxidases, NOR derives its substrate protons from the periplasmic (membrane potential-positive) side of the membrane. The molecular details of this non-electrogenic proton transfer are not yet resolved, so in this study we have explored a role in a proposed proton pathway for a conserved surface glutamate (Glu-122) in the catalytic subunit (NorB). The effect of substituting Glu-122 with Ala, Gln, or Asp on a single turnover of the reduced NOR variants with O(2), an alternative and experimentally tractable substrate for NOR, was determined. Electron transfer coupled to proton uptake to the bound O(2) is severely and specifically inhibited in both the E122A and E122Q variants, establishing the importance of a protonatable side chain at this position. In the E122D mutant, proton uptake is retained but it is associated with a significant increase in the observed pK(a) of the group donating protons to the active site. This suggests that Glu-122 is important in defining this proton donor. A second nearby glutamate (Glu-125) is also required for the electron transfer coupled to proton uptake, further emphasizing the importance of this region of NorB in proton transfer. Because Glu-122 is predicted to lie near the periplasmic surface of NOR, the results provide strong experimental evidence that this residue contributes to defining the aperture of a non-electrogenic "E-pathway" that serves to deliver protons from the periplasm to the buried active site in NOR.

We have investigated the kinetics of the single-turnover reaction of fully reduced solubilised cytochrome c oxidase (cytochrome aa3) from Rhodobacter sphaeroides with dioxygen using the flow-flash methodology and compared the results to those obtained with the well-characterised bovine mitochondrial enzyme. The overall reaction sequence was the same in the two enzymes, but the extents and rates of the electron-transfer reactions differed, implying differences in redox potentials, and/or interaction energies between electrons and protons during oxygen reduction. As with the bovine enzyme, the R. sphaeroides enzyme displayed two major kinetic phases of proton uptake with rate constants of approximately 5000 s-1 and approximately 500 s-1 at pH 7.9, concomitant with the peroxy to oxoferryl and oxoferryl to oxidised states. The net number of protons taken up in the R. sphaeroides enzyme was about approximately 1.9, which implies that upon reduction, the enzyme has to pick up approximately 2.1 H+ from the medium. On the basis of the comparison of electron-transfer reactions in the two enzymes, we conclude that the transfer rate of the fourth electron to the binuclear centre is not only determined by the electron-transfer rate from haem a to the binuclear centre, but also by the electron equilibrium between CuA and haem a. In addition, in contrast to the bovine enzyme, where the electron- and proton-transfer rates during oxidation of the fully reduced enzyme by O2 are all faster than the overall turnover rate, in the R. sphaeroides enzyme, the slowest kinetic phase was rate limiting for the overall turnover. Moreover, the comparison of the reactions in the two systems shows that in the R. sphaeroides enzyme, the electrons are more evenly distributed among the redox centres during oxygen reduction. This enables investigations of effects also of minor perturbations on, e.g., the electron-transfer characteristics in mutant enzymes, for which this study forms the basis.

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase from Bacillus PS3 in the presence of O2 has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochrome c subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 microseconds, 30 microseconds, 100 microseconds, and I ms at 445 nm. The initial, 10-microsecond absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving heme a and CuA are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochrome c, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O2 were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from heme a3 to a in PS3 oxidase compared to the bovine enzyme.

Heme-copper oxidases (HCuOs) are integral membrane proteins that catalyze the reduction of oxygen. They terminate the respiratory chain in mitochondria and most bacteria. HCuOs convert the free energy of oxygen reduction into a proton-motive force across the membrane, by pumping protons over the membrane and by using protons from the negative side of the membrane while electrons are donated from the positive side. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR). Even though NORs are capable of reducing oxygen to water, their physiological roles are as NO reducers. NO reduction is performed by denitrifying bacteria, but also by pathogens that use it to break down the toxic NO produced by the immune system of the host.In the NORs, recent structural work has shown that there are possible differences in the proton transfer (PT) pathways of the NOR that uses cyt. c as electron donor (cNOR) and the NOR that uses quinol (qNOR). For qNOR a putative PT pathway from the cytoplasm was suggested, while cNOR is known to be non-electrogenic and to use periplasmic protons for NO-reduction (2NO + 2e− + 2H+ → N2O + H2O). We study the PT pathways using site-directed mutagenesis in combination with time-resolved spectroscopy during single turnovers. We show that PT in cNOR is mediated by a specific pathway from the periplasmic site. At the same time we try to engineer a proton transfer pathway from the cytoplasm in cNOR to see if we can generate an electrogenic NOR and to get more insight into the requirements for a functional PT pathway.

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g 12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging car☐yl ligand in the bimetallic cytochrome a 3 -Cu B site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the car☐yl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a , and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a . These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.

Heme–copper oxidases (HCuOs) are the terminal components of the respiratory chain in the mitochondrial membrane or the cell membrane in many bacteria. These enzymes reduce oxygen to water and use the free energy from this reaction to maintain a proton-motive force across the membrane in which they are embedded. The heme–copper oxidases of the cbb3-type are only found in bacteria, often pathogenic ones since they have a low Km for O2, enabling the bacteria to colonize semi-anoxic environments. Cbb3-type (C) oxidases are highly divergent from the mitochondrial-like aa3-type (A) oxidases, and within the heme–copper oxidase family, cbb3 is the closest relative to the most divergent member, the bacterial nitric oxide reductase (NOR). Nitric oxide reductases reduce NO to N2O without coupling the reaction to the generation of any electrochemical proton gradient. The significant structural differences between A- and C-type heme–copper oxidases are manifested in the lack in cbb3 of most of the amino acids found to be important for proton pumping in the A-type, as well as in the different binding characteristics of ligands such as CO, O2 and NO. Investigations of the reasons for these differences at a molecular level have provided insights into the mechanism of O2 and NO reduction as well as the proton-pumping mechanism in all heme–copper oxidases. In this paper, we discuss results from these studies with the focus on the relationship between proton transfer and ligand binding and reduction. In addition, we present new data, which show that CO binding to one of the c-type hemes of CcoP is modulated by protein–lipid interactions in the membrane. These results show that the heme c-CO binding can be used as a probe of protein–membrane interactions in cbb3 oxidases, and possible physiological consequences for this behavior are discussed.