Aerobic organisms obtain energy by linking electron transfer from NADH to O₂, through the respiratory chain, to transmembrane proton translocation. In mycobacteria the respiratory chain is branched; the membrane-bound electron carrier menaquinol (MQH₂) donates electrons either to the O₂-reducing cytochrome bd or a supercomplex that is composed of a complex (C) III₂ dimer flanked by two CIVs. Here, we measured the dimethyl-naphthoquinone (DMNQH_2, a menaquinol analogue) oxidation:O₂ reduction activities of the CIII₂CIV₂ supercomplex and cytochrome bd in the presence of an analogue (decylubiquinone, DCQ) of the mammalian electron carrier, ubiquinol. The data show that DCQH₂ inhibits both the CIII₂CIV₂ and cytochrome bd activities, suggesting that DCQ/DCQH₂ interferes with both branches of the respiratory chain. Cryo-EM data of the M. smegmatis supercomplex shows that oxidized DCQ binds in the electron donor site (Q_o) of CIII₂. Accordingly, growth of M. smegmatis cells was impaired in the presence of DCQ. Remarkably, DCQ also impairs intracellular growth of virulent M. tuberculosis cells in human primary macrophages suggesting that the compound could potentially be used as an adjuvant during tuberculosis disease treatment.

ATPases associated with diverse cellular activities (AAA+ -ATPases) catalyse a wide range of remodelling events in all phyla. AAA+ -ATPases of the MoxR-like family typically co-operate with von Willebrand factor type A (VWA) domain containing proteins to facilitate target remodelling and metal ion insertion, but their mechanism of action is poorly understood. We studied the bacterial AAA+ -ATPase NorQ in complex with its VWA domain partner protein NorD, which are essential for nitric oxide reductase (NOR) activity. Our cryo-EM structures and biochemical analyses show that NorQ and NorD engage through two key interfaces: (i) a finger-like extension protruding from the VWA domain that penetrates the central pore of the NorQ hexamer, and (ii) the NorD C- terminus, which contacts the post-sensor 1 loop of NorQ. Our data reveal that NorQ activity remodels a linker region in NorD essential for metal insertion. Together, these findings support a model in which the NorQ complex exerts a twisting and stretching force on the NorD linker, thereby enabling metal insertion into its target NOR.

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here, we characterize the cyt bd-II from Mycobacterium smegmatis, a member of a hitherto uncharacterized evolutionary group (qOR-2) of bd oxidases, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. Overexpressing the appCB operon in its native host led to production of a highly active bd-II (k_obs = 30 e⁻ s⁻¹) that together with a high-resolution (2.8 Å) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

Cytochrome c-dependent nitric oxide reductase (cNOR) catalyzes the reduction of NO into nitrous oxide (N₂O), a strong greenhouse gas released from denitrifying microorganisms. The cNOR active site holds an essential non-heme iron, Fe_B, inserted using the chaperone complex NorQD. However, in Thermus thermophilus, the cNOR (TtcNOR) cluster lacks the norQD genes. Here we investigated Fe_B insertion into TtcNOR and characterized and compared TtcNOR expressed in Escherichia coli to that natively produced. We show that Fe_B is present in the natively produced TtcNOR only. Analysis of cNOR operon sequences suggests that a hydrophilic K-pathway analogue is present in cNORs that do not rely on NorQD for iron insertion. We discuss the implications of our data for the evolution of the NOR family.

Nitrous oxide (N₂O) is a serious concern due to its role in global warming and ozone destruction. Agricultural practices account for ∼80% of all anthropogenic N₂O produced in the US, due in large part to the stimulation of microbial denitrification. Stable isotopes are uniquely suited to examine both microbial N₂O sources and the mechanism of N₂O biosynthesis through the use of Site Preference (δ¹⁵N^SP; the difference in δ¹⁵N between the central and outer N atoms in N₂O) and kinetic isotope effects (KIEs), respectively. Using trace gas isotope ratio mass spectrometry (TG-IRMS), we determined the δ¹⁵N, δ¹⁵N^α, δ¹⁵N^β, and δ¹⁸O of N₂O produced by a purified cytochrome c nitric oxide reductase (cNOR) from Paracoccus denitrificans. We also calculated δ¹⁵N^SP, the KIEs, and associated isotopic enrichment factors (ε) for N^bulk, N^α, and N^β. A normal isotope effect was observed for bulk ¹⁵N, with a KIE value of 1.0086 ± 0.0009 (ε = −8.6 ± 0.9‰). The isotope effects for both ¹⁵N^α and ¹⁵N^β were also normal, with position-specific KIEs of 1.0072 ± 0.0010 (ε = −7.2 ± 1.0‰) and 1.0100 ± 0.0010 (ε = −9.9 ± 1.0‰), respectively, and δ¹⁵N^SP values ranged from 0.5 to 8.7‰ with no significant trend as the reaction proceeded. Values of δ¹⁸O increased with N₂O production (slope of δ¹⁸O against [−f ln f/(1 – f)] = −19.9 ± 1.9‰). We present implications for the mechanism of N₂O production from cNOR based on our data.

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.

Cytochrome bds are bacterial terminal oxidases expressed under low oxygen conditions, and they are important for the survival of many pathogens and hence potential drug targets. The largest subunit CydA contains the three redox-active cofactors heme b558, heme b595 and the active site heme d. One suggested proton transfer pathway is found at the interface between the CydA and the other major subunit CydB. Here we have studied the O2 reduction mechanism in E. coli cyt. bd-I using the flow-flash technique and focused on the mechanism, kinetics and pathway for proton transfer. Our results show that the peroxy (P) to ferryl (F) transition, coupled to the oxidation of the low-spin heme b558 is pH dependent, with a maximum rate constant (~104 s−1) that is slowed down at higher pH. We assign this behavior to rate-limitation by internal proton transfer from a titratable residue with pKa ~ 9.7. Proton uptake from solution occurs with the same P➔F rate constant. Site-directed mutagenesis shows significant effects on catalytic turnover in the CydB variants Asp58B➔Asn and Asp105B➔Asn variants consistent with them playing a role in proton transfer. Furthermore, in the Asp105B➔Asn variant, the reactions up to P formation occur essentially as in the wildtype bd-I, but the P➔F transition is specifically inhibited, supporting a direct and specific role for Asp105B in the functional proton transfer pathway in bd-I. We further discuss the possible identity of the high pKa proton donor, and the conservation pattern of the Asp-105B in the cyt. bd superfamily.

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.

Fission yeast Schizosaccharomyces pombe serves as model organism for studying higher eukaryotes. We combined the use of cryo-EM and spectroscopy to investigate the structure and function of affinity purified respiratory complex IV (CIV) from S. pombe. The reaction sequence of the reduced enzyme with O-2 proceeds over a time scale of mu s-ms, similar to that of the mammalian CIV. The cryo-EM structure of CIV revealed eleven subunits as well as a bound hypoxia-induced gene 1 (Hig1) domain of respiratory supercomplex factor 2 (Rcf2). These results suggest that binding of Rcf2 does not require the presence of a CIII-CIV supercomplex, i.e. Rcf2 is a component of CIV. An AlphaFold-Multimer model suggests that the Hig1 domains of both Rcf1 and Rcf2 bind at the same site of CIV suggesting that their binding is mutually exclusive. Furthermore, the differential functional effect of Rcf1 or Rcf2 is presumably caused by interactions of CIV with their different non-Hig1 domain parts. Fission yeast Schizosaccharomyces pombe shares many characteristics with higher eukaryotes. Here, the authors investigate the structure and function of respiratory complex IV from S. pombe, reveal the subunit arrangements and the reaction sequence of O-2 reduction.

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 (Fe_B) 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 Fe_B 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.