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The family of flavodiiron proteins (FDPs) plays an important role in the scavenging and detoxification of both molecular oxygen and nitric oxide. Using electrons from a flavin mononucleotide cofactor molecular oxygen is reduced to water and nitric oxide is reduced to nitrous oxide and water. While the mechanism for NO reduction in FDPs has been studied extensively, there is very little information available about O 2 reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O 2 reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O 2 molecule has bound to the diferrous diiron active site and before the O O bond can be cleaved. This is in contrast to the mechanism for NO reduction, where both N N bond formation and N O bond cleavage occurs from the same starting structure without any further reduction, according to both experimental and computational results. This computational result for the O 2 reduction mechanism should be possible to evaluate experimentally. Another difference between the two substrates is that the actual O O bond cleavage barrier is low, and not involved in rate-limiting the reduction process, while the barrier connected with bond cleavage/formation in the NO reduction process is of similar height as the rate-limiting steps. We suggest that these results may be part of the explanation for the generally higher activity for O 2 reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O 2 reduction reaction in the family of heme‑copper oxidases. • We suggest a full mechanism for O 2 reduction to water in Flavodiiron Proteins. • Energy profile for a full catalytic cycle for O 2 reduction is constructed. • Proton coupled reduction occurs before the O O bond can be cleaved. • We provide explanations for differences in reactivity between O 2 and NO. • Comparisons to Heme-Copper-Oxidases provide new insights. [ABSTRACT FROM AUTHOR]

Abstract Cytochrome c oxidases (C c O) reduce O 2 to H 2 O in the respiratory chain of mitochondria and many aerobic bacteria. In addition, some species of C c O can also reduce NO to N 2 O and water while others cannot. Here, the mechanism for NO-reduction in C c O is investigated using quantum mechanical calculations. Comparison is made to the corresponding reaction in a "true" cytochrome c -dependent NO reductase (c NOR). The calculations show that in c NOR, where the reduction potentials are low, the toxic NO molecules are rapidly reduced, while the higher reduction potentials in C c O 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 c NOR, N 2 O can then be formed using only the active-site electrons. In contrast, in C c O, one proton-coupled reduction step most likely has to occur before N 2 O can be formed, and furthermore, proton transfer is most likely rate-limiting. This can explain why different C c O species with the same heme a 3 -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 C c O. Graphical Abstract Highlights • Mechanisms for NO reduction in C c Os with a heme a 3 active site are studied. • Free energy profiles are constructed combining computational and experimental data. • Comparisons are made to the mechanism for NO reduction in cNOR. • High reduction potentials in C c Os are found to cause low rates of NO disappearance. • A conserved valine may play a role in reducing the NO reduction rate in C c O. [ABSTRACT FROM AUTHOR]

Heme-copper oxidase (HCO) is a class of respiratory enzymes that use a heme-copper center to catalyze O2 reduction to H2O. While heme reduction potential (E°′) of different HCO types has been found to vary >500 mV, its impact on HCO activity remains poorly understood. Here, we use a set of myoglobin-based functional HCO models to investigate the mechanism by which heme E°′ modulates oxidase activity. Rapid stopped-flow kinetic measurements show that increasing heme E°′ by ca. 210 mV results in increases in electron transfer (ET) rates by 30-fold, rate of O2 binding by 12-fold, O2 dissociation by 35-fold, while decreasing O2 affinity by 3-fold. Theoretical calculations reveal that E°′ modulation has significant implications on electronic charge of both heme iron and O2, resulting in increased O2 dissociation and reduced O2 affinity at high E°′ values. Overall, this work suggests that fine-tuning E°′ in HCOs and other heme enzymes can modulate their substrate affinity, ET rate and enzymatic activity. [ABSTRACT FROM AUTHOR]

Abstract: Heme-copper oxidases (HCuOs) catalyse the reduction of oxygen, using the liberated free energy to maintain a proton-motive force across the membrane. In the mitochondrial-like A-type HCuOs, binding of heavy metal ions at the surface of the protein inhibits proton transfer. In bacterial C-type oxidases, the entry point to the proton pathway is on an accessory subunit unrelated to any subunit in A-type HCuOs. Despite this, we show here that heavy metal ions such as Zn2+ inhibit O2-reduction very similarly in C-type as in A-type HCuOs, and furthermore that the binding site shares the same Glu-His motif. [Copyright &y& Elsevier]

Heme-copper oxidases (HCuOs) are the last components of the respiratory chain in mitochondria and many bacteria. They catalyze O2 reduction and couple it to the maintenance of a proton-motive force across the membrane in which they are embedded. In the mitochondrial-like, A family of HCuOs, there are two well established proton transfer pathways leading from the cytosol to the active site, the D and the K pathways. In the C family (cbb3) HCuOs, recent work indicated the use of only one pathway, analogous to the K pathway. In this work, we have studied the functional importance of the suggested entry point of this pathway, the Glu-25 (Rhodobacter sphaeroides cbb3 numbering) in the accessory subunit CcoP (E25P). We show that catalytic turnover is severely slowed in variants lacking the protonatable Glu-25. Furthermore, proton uptake from solution during oxidation of the fully reduced cbb3 by O2 is specifically and severely impaired when Glu-25 was exchanged for Ala or Gln, with rate constants 100-500 times slower than in wild type. Thus, our results support the role of E25P as the entry point to the proton pathway in cbb3 and that this pathway is the main proton pathway. This is in contrast to the A-type HCuOs, where the D (and not the K) pathway is used during O2 reduction. The cbb3 is in addition to O2 reduction capable of NO reduction, an activity that was largely retained in the E25P variants, consistent with a scenario where NO reduction in cbb3 uses protons from the periplasmic side of the membrane. [ABSTRACT FROM AUTHOR]

Energy conservation in all kingdoms of life involves electron transfer, through a number of membrane-bound proteins, associated with proton transfer across the membrane. In aerobic organisms, the last component of this electron-transfer chain is a respiratory heme-copper oxidase that catalyzes reduction of O2 to H2O, linking this process to transmembrane proton pumping. So far, the molecular mechanism of proton pumping is not known for any system that is driven by electron transfer. Here, we show that this problem can be addressed and elucidated in a unique cytochrome c oxidase (cytochrome ba3) from a thermophilic bacterium, Thermus thermophilus. The results show that in this oxidase the electron- and proton-transfer reactions are orchestrated in time such that previously unresolved proton-transfer reactions could be directly observed. On the basis of these data we propose that loading of the proton pump occurs upon electron transfer, but before substrate proton transfer, to the catalytic site. Furthermore, the results suggest that the pump site alternates between a protonated and deprotonated state for every second electron transferred to the catalytic site, which would explain the noninteger pumping stoichiometry (0.5 H+/e-) of the ba3 oxidase. Our studies of this variant of Nature's palette of mechanistic solutions to a basic problem offer a route toward understanding energy conservation in biological systems. [ABSTRACT FROM AUTHOR]

The heme-copper oxidase (HCuO) superfamily consists of integral membrane proteins that catalyze the reduction of either oxygen or nitric oxide. The HCuOs that reduce O2 to H2O couple this reaction to the generation of a transmembrane proton gradient by using electrons and protons from opposite sides of the membrane and by pumping protons from inside the cell or organelle to the outside. The bacterial NO-reductases (NOR) reduce NO to N2O (2NO + 2e- + 2H+ → N2O + H2O), a reaction as exergonic as that with O2. Yet, in NOR both electrons and protons are taken from the outside periplasmic solution, thus not conserving the free energy available. The cbb3-type HCuOs catalyze reduction of both O2 and NO. Here, we have investigated energy conservation in the Rhodobacter sphaeroides cbb3 oxidase during reduction of either O2 or NO. Whereas O2 reduction is coupled to buildup of a substantial electrochemical gradient across the membrane, NO reduction is not. This means that although the cbb3 oxidase has all of the structural elements for uptake of substrate protons from the inside, as well as for proton pumping, during NO reduction no pumping occurs and we suggest a scenario where substrate protons are derived from the outside solution. This would occur by a reversal of the proton pathway normally used for release of pumped protons. The consequences of our results for the general pumping mechanism in all HCuOs are discussed. [ABSTRACT FROM AUTHOR]

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons. [ABSTRACT FROM AUTHOR]

Abstract 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 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 p K 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. Graphical abstract Unlabelled Image Highlights • We studied variants with the KC proton pathway modified; Y227F, N293L and Y321F. • In N293L, proton-coupled electron transfer is slowed. • Further, in N293L we could observe titration of an internal proton door XH. • In Y321F, proton uptake from bulk solution is greatly impaired. • We suggest that Y321 is among the candidates for the important proton donor XH. [ABSTRACT FROM AUTHOR]

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 integralmembrane 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 periplasminto 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. [ABSTRACT FROM AUTHOR]

Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O2 to H2O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba3 oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O2 reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H+/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1?H+/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba3 oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a "proton-loading site" (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 cbb 3-type are only found in bacteria, often pathogenic ones since they have a low K m for O2, enabling the bacteria to colonize semi-anoxic environments. Cbb 3-type (C) oxidases are highly divergent from the mitochondrial-like aa 3-type (A) oxidases, and within the heme–copper oxidase family, cbb 3 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 cbb 3 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 cbb 3 oxidases, and possible physiological consequences for this behavior are discussed. [Copyright &y& Elsevier]