Flavodiiron proteins (FDPs) constitute a large family of non-heme iron enzymes present in all domains of life. They play important roles as scavengers and detoxifiers by efficiently reducing both O₂ and NO. The primary ligands of the diiron active site in all FDPs are highly conserved, indicating that the basic reaction mechanisms for O₂ and NO reduction, respectively, are the same. However, the reduction activity varies significantly between different FDPs. By comparing FDPs from two different species, Thermotoga maritima and Desulfovibrio gigas, we investigate to what extent variations in the second sphere ligation can explain differences in reduction activities. Comparisons are also made between wildtype and two variants of Thermotoga maritima FDP. We use Density functional theory (DFT) calculations on a number of FDP active site models to study the reaction mechanisms for both O₂ and NO reduction. For reduction of O₂ we conclude that differences in activity cannot be explained by differences in the first or second active site coordination spheres, which is mainly due to a low barrier for OO bond cleavage after one proton-coupled reduction step. For NO reduction however, the rate-limiting barrier for N₂O formation, a hyponitrite rotation, is high enough to be involved in the overall rate limitation. We show that second sphere residues, such as Tyr26 in Desulfovibrio gigas FDP, that can form hydrogen bonds to the rotating hyponitrite, decrease the barrier. Differences in NO reduction rate among different FDPs are most likely determined by the variation in such second sphere residues.

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 O₂ reduction. Here we use hybrid density functional theory (DFT) to study the mechanism for O₂ reduction in FDPs. An important finding is that a proton coupled reduction is needed after the O₂ 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 O₂ 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 O₂ reduction as compared to NO reduction in most FDPs. Comparisons are also made to the O₂ reduction reaction in the family of heme‑copper oxidases.

The flavin dependent nonheme diiron proteins comprise a family of enzymes, which can act as scavengers for both molecular oxygen and nitric oxide. The reduction of nitric oxide to nitrous oxide and water in flavodiiron proteins (FDPs) has been studied both experimentally and computationally, but the reaction mechanism is far from well understood. From experiments, it is known that two NO molecules can bind to the reduced active site, forming an observable diferrous dinitrosyl complex. A main question has been whether nitrous oxide can be formed directly from the diferrous dinitrosyl complex or if further reduction and/or protonation is needed to make this step feasible. Experiments have shown that nitrous oxide can be formed in a deflavinated form of the enzyme, indicating that further reduction is not needed. In the present study, hybrid density functional theory calculations are performed on a cluster model of the Thermotoga maritima FDP active site. We show that nitric oxide can be reduced to nitrous oxide and water using a direct coupling mechanism, i.e., without further additions to the reduced active site. The diferrous dinitrosyl complex can form an unstable N-N bridging hyponitrite intermediate, which can rotate into an N-O bond bridging hyponitrite with a low barrier. From this intermediate, the N-O bond cleavage leading to release of nitrous oxide is energetically feasible. An energy profile for the entire catalytic cycle of such a direct coupling mechanism is presented, and it is shown that the suggested mechanism agrees with data on FDP variants. Finally, an energy profile for the entire process starting with the fully reduced enzyme turning over four NO equivalents is constructed. This energy profile suggests explanations to experimentally observed states, such as the dihydroxyl form of the fully oxidized diferric state, and the difference with respect to returning to the original oxidized state after NO reduction between the flavinated and the deflavinated form of the enzyme.

Significant improvements of the density functional theory (DFT) methodology during the past few decades have made DFT calculations a powerful tool in studies of enzymatic reaction mechanisms. For metalloenzymes, however, there are still concerns about the reliability in the DFT-results. Therefore, a systematic study is performed where the fraction of exact exchange in a hybrid DFT functional is used as a parameter. By varying this parameter, a set of different but related functionals are obtained. The various functionals are applied to one of the reactions occurring in the enzyme family heme–copper oxidases, the reduction of nitric oxide (NO) to nitrous oxide (N₂O) and water. The results show that, even though certain parts of the calculated energetics exhibit large variations, the qualitative pictures of the reaction mechanisms are quite stable. Furthermore, it is found that the functional with 15% exact exchange (B3LYP^*) gives the best agreement with experimental data for the particular reactions studied. An important aspect of the procedure used is that the computational results are carefully combined with a few more general experimental data to obtain a complete description of the entire catalytic cycle of the reactions studied.

Cellular respiration involves electron transport via a number of enzyme complexes to the terminal Cytochrome c oxidase (CcO), in which molecular oxygen is reduced to water. The free energy released in the reduction process is used to establish a transmembrane electrochemical gradient, via two processes, both corresponding to charge transport across the membrane in which the enzymes are embedded. First, the reduction chemistry occurring in the active site of CcO is electrogenic, which means that the electrons and protons are delivered from opposite sides of the membrane. Second, the exergonic chemistry is coupled to translocation of protons across the entire membrane, referred to as proton pumping. In the largest subfamily of the CcO enzymes, the A-family, one proton is pumped for every electron needed for the chemistry, making the energy conservation particularly efficient. In the present study, hybrid density functional calculations are performed on a model of the A-family CcOs. The calculations show that the redox-active tyrosine, conserved in all types of CcOs, plays an essential role for the energy conservation. Based on the calculations a reaction mechanism is suggested involving a tyrosyl radical (possibly mixed with tyrosinate character) in all reduction steps. The result is that the free energy released in each reduction step is large enough to allow proton pumping in all reduction steps without prohibitively high barriers when the gradient is present. Furthermore, the unprotonated tyrosine provides a mechanism for coupling the uptake of two protons per electron in every reduction step, i.e. for a secure proton pumping.

Heme-copper oxidases are transmembrane enzymes involved in aerobic and anaerobic respiration. The largest subgroup contains the cytochromecoxidases (CcO), which reduce molecular oxygen to water. A significant part of the free energy released in this exergonic process is conserved as an electrochemical gradient across the membrane,viatwo processes, electrogenic chemistry and proton pumping. A deviant subgroup is the cytochromecdependent NO reductases (cNOR), which reduce nitric oxide to nitrous oxide and water. This is also an exergonic reaction, but in this case none of the released free energy is conserved. Computational studies applying hybrid density functional theory to cluster models of the bimetallic active sites in the heme-copper oxidases are reviewed. To obtain a reliable description of the reaction mechanisms, energy profiles of the entire catalytic cycles, including the reduction steps have to be constructed. This requires a careful combination of computational results with certain experimental data. Computational studies have elucidated mechanistic details of the chemical parts of the reactions, involving cleavage and formation of covalent bonds, which have not been obtainable from pure experimental investigations. Important insights regarding the mechanisms of energy conservation have also been gained. The computational studies show that the reduction potentials of the active site cofactors in the CcOs are large enough to afford electrogenic chemistry and proton pumping,i.e.efficient energy conservation. These results solve a conflict between different types of experimental data. A mechanism for the proton pumping, involving a specific and crucial role for the active site tyrosine, conserved in all CcOs, is suggested. For thecNORs, the calculations show that the low reduction potentials of the active site cofactors are optimized for fast elimination of the toxic NO molecules. At the same time, the low reduction potentials lead to endergonic reduction steps with high barriers. To prevent even higher barriers, which would lead to a too slow reaction, when the electrochemical gradient across the membrane is present, the chemistry must occur in a non-electrogenic manner. This explains why there is no energy conservation incNOR.

The superfamily of heme copper oxidases reduces molecular oxygen or nitric oxide, and the active sites comprise a high-spin heme group (a(3) or b(3)) and a non-heme metal (Cu-B or Fe-B). The cbb(3) C family of cytochrome c oxidases, with the highspin heme b(3) and CUB in the active site, is a subfamily of the heme copper oxidases that can reduce both molecular oxygen, which is the main substrate, and nitric oxide. The mechanism for NO reduction in cbb(3) oxidase is studied here using hybrid density functional theory and compared to other cytochrome c oxidases (A and B families), with a high-spin heme a(3) and Cu-B in the active site, and to cytochrome c dependent NO reductase, with a high-spin heme b(3) and a nonheme Fe-B in the active site. It is found that the reaction mechanism and the detailed reaction energetics of the cbb(3) oxidases are not similar to those of cytochrome c dependent NO reductase, which has the same type of high-spin heme group but a different nonheme metal. This is in contrast to earlier expectations. Instead, the NO reduction mechanism in cbb(3) oxidases is very similar to that in the other cytochrome c oxidases, with the same non-heme metal, CUB, and is independent of the type of high-spin heme group. The conclusion is that the type of non-heme metal (CUB or Fe-B) in the active site of the heme copper oxidases is more important for the reaction mechanisms than the type of high-spin heme, at least for the NO reduction reaction. The reason is that the protoncoupled reduction potentials of the active site cofactors determine the energetics for the NO reduction reaction, and they depend to a larger extent on the non-heme metal. Observed differences in NO reduction reactivity among the various cytochrome c oxidases may be explained by differences outside the BNC, affecting the rate of proton transfer, rather than in the BNC itself.

Cytochrome c oxidases (CcOs) couple the exergonic reduction of molecular oxygen to proton pumping across the membrane in which they are embedded, thereby conserving a significant part of the free energy. The A family CcOs are known to pump four protons per oxygen molecule, while there is no consensus regarding the proton pumping stoichiometry for the C family cbb(3) oxidases. Hybrid density functional theory is used here to investigate the catalytic mechanism for oxygen reduction in cbb(3) oxidases. A surprising result is that the barrier for O-O bond cleavage at the mixed valence reduction level seems to be too high compared to the overall reaction rate of the enzyme. It is therefore suggested that the O-O bond is cleaved only after the first proton coupled reduction step, and that this reduction step most likely is not coupled to proton pumping. Furthermore, since the cbb3 oxidases have only one proton channel leading to the active site, it is proposed that the activated E-H intermediate, suggested to be responsible for proton pumping in one of the reduction steps in the A family, cannot be involved in the catalytic cycle for cbb(3), which results in the lack of proton pumping also in the E to R reduction step. In summary, the calculations indicate that only two protons are pumped per oxygen molecule in cbb(3) oxidases. However, more experimental information on this divergent enzyme is needed, e.g. whether the flow of electrons resembles that in the other more well-studied CcO families.

Cytochrome c oxidase (CcO), the terminal enzyme in the respiratory chain, reduces molecular oxygen to water. Experimental data on the midpoint potentials of the heme iron/copper active site cofactors do not match the overall reaction energetics, and are also in conflict with the observed efficiency of energy conservation in CcO. Therefore it has been postulated that the ferric/cupric intermediate (the oxidized state) exists in two forms. One form, labelled O-H, is presumably involved during catalytic turnover, and should have a high Cu-B midpoint potential due to a metastable high energy structure. When no more electrons are supplied, the O-H state supposedly relaxes to the resting form, labelled O, with a lower energy and a lower midpoint potential. It has been suggested that there is a pure geometrical difference between the O-H and O states, obtained by moving a water molecule inside the active site. It is shown here that the difference between the two forms of the oxidized state must be of a more chemical nature. The reason is that all types of geometrically relaxed structures of the oxidized intermediate have similar energies, all with a high proton coupled reduction potential in accordance with the postulated O-H state. One hypothesized chemical modification of the O-H state is the transfer of an extra proton, possibly internal, into the active site. Such a protonated state has several properties that agree with experimental data on the relaxed oxidized state, including a decreased midpoint potential.

The isotopic composition of nitrous oxide (N2O) provides useful information for evaluating N2O sources and budgets. Due to the co-occurrence of multiple N2O transformation pathways, it is, however, challenging to use isotopic information to quantify the contribution of distinct processes across variable spatiotemporal scales. Here, we present an overview of recent progress in N2O isotopic studies and provide suggestions for future research, mainly focusing on: analytical techniques; production and consumption processes; and interpretation and modelling approaches. Comparing isotope-ratio mass spectrometry (IRMS) with laser absorption spectroscopy (LAS), we conclude that IRMS is a precise technique for laboratory analysis of N2O isotopes, while LAS is more suitable forin situ/inline studies and offers advantages for site-specific analyses. When reviewing the link between the N2O isotopic composition and underlying mechanisms/processes, we find that, at the molecular scale, the specific enzymes and mechanisms involved determine isotopic fractionation effects. In contrast, at plot-to-global scales, mixing of N2O derived from different processes and their isotopic variability must be considered. We also find that dual isotope plots are effective for semi-quantitative attribution of co-occurring N2O production and reduction processes. More recently, process-based N2O isotopic models have been developed for natural abundance and(15)N-tracing studies, and have been shown to be effective, particularly for data with adequate temporal resolution. Despite the significant progress made over the last decade, there is still great need and potential for future work, including development of analytical techniques, reference materials and inter-laboratory comparisons, further exploration of N2O formation and destruction mechanisms, more observations across scales, and design and validation of interpretation and modelling approaches. Synthesizing all these efforts, we are confident that the N2O isotope community will continue to advance our understanding of N2O transformation processes in all spheres of the Earth, and in turn to gain improved constraints on regional and global budgets.