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NifB is a radical S -adenosyl-L-methionine (SAM) enzyme that is essential for nitrogenase cofactor assembly. Previously, a nitrogen ligand was shown to be involved in coupling a pair of [Fe 4 S 4 ] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe 8 S 9 C] cofactor core (designated L) on NifB. However, the identity and function of this ligand remain elusive. Here, we use combined mutagenesis and pulse electron paramagnetic resonance analyses to establish histidine-43 of Methanosarcina acetivorans NifB ( Ma NifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of Ma NifB to serve as a source for cofactor maturation upon substitution of histidine-43 with alanine; whereas x-ray absorption spectroscopy/extended x-ray fine structure experiments further suggest formation of an intermediate that lacks the cofactor core arrangement in this Ma NifB variant. These results point to dual functions of histidine-43 in structurally assisting the proper coupling between K1 and K2 and concurrently facilitating carbide formation via deprotonation of the initial carbon radical. NifB is a radical SAM enzyme involved in the biosynthesis of the Mo-nitrogenase cofactor, which is responsible for the ambient conversion of N 2 to NH 3 . Here, the authors identify and uncover the function of a His43 residue as an essential nitrogen ligand of NifB in cofactor biosynthesis.

NifB is an essential radical S -adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe 4 S 4 ] modules (designated K1 and K2) into an [Fe 8 S 9 C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe 4 S 4 ] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe 4 S 4 ] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5′-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron–sulfur (FeS) enzymes. NifB is a key enzyme in the biosynthesis pathway of the nitrogenase FeMo cofactor. Here, the authors investigate the maturation of its iron-sulfur clusters by EPR and biochemical analyses, showing how individual precursor clusters participate in the formation of the final iron-sulfur cluster.

Molybdenum nitrogenase catalyses the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the ‘ninth sulfur’ in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon ‘ninth sulfur’ insertion remain elusive. Here we trace the ‘ninth sulfur’ insertion using SeO32− and TeO32− as ‘labelled’ SO32−. Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the ‘ninth sulfur’ in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2−- and Te2−-like species into the L-cluster. Density functional theory calculations further point to a plausible mechanism involving in situ reduction of SO32− to S2−, thereby suggesting the utility of this reaction to label the catalytically important belt region for mechanistic investigations of nitrogenase.Located in the catalytically important belt region, the ‘ninth sulfur’ of the nitrogenase cofactor has now been shown to be inserted through coordination of sulfite by two cluster iron atoms at a vacant belt site. This is followed by in situ reduction of sulfite to sulfide, which enables the subsequent transfer and functionalization of the cofactor.

We review here the recent literature dealing with the molybdenum- and copper-dependent CO dehydrogenase, with particular emphasis on the structure of the enzyme and recent advances in our understanding of the reaction mechanism of the enzyme.

Nitrate reductases play pivotal roles in nitrogen metabolism by leveraging the molybdopterin cofactor to facilitate the reduction of nitrate to nitrite. Periplasmic nitrate reductases (NapA) utilize nitrate as a terminal electron acceptor when oxygen is limiting, helping to drive anaerobic metabolism in bacteria. Despite extensive research into NapA homologs, open questions about the mechanism remain especially at the molecular level. More broadly, little is understood of how the molybdopterin cofactor is tuned for catalysis in these enzymes enabling broad substrate scope and reactivity observed in molybdenum-containing enzymes. Here, we have prepared NapA from Campylobacter jejuni under single turnover conditions to generate a singly reduced enzyme that can be further examined by electron paramagnetic resonance (EPR) spectroscopy. Our results provide new context into the known spectra and related structures of NapA and related enzymes. These insights open new avenues for understanding nitrate reductase mechanisms, molybdenum coordination dynamics, and the role of pyranopterin ligands in catalysis.

Periplasmic nitrate reductase NapA from Campylobacter jejuni ( C. jejuni ) contains a molybdenum cofactor (Moco) and a 4Fe–4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe–4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe–4S cluster. K79 forms H-bonding interactions with the 4Fe–4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed. Graphical abstract

NifB is an essential radical S-adenosylmethionine (SAM) enzyme for nitrogenase cofactor assembly. Previous studies show that NifB couples a putative pair of [Fe S ] modules (designated K1 and K2) into an [Fe S C] cofactor precursor concomitant with radical SAM-dependent carbide insertion through the action of its SAM-binding [Fe S ] module. However, the coordination and function of the NifB cluster modules remain unknown. Here, we use continuous wave and pulse electron paramagnetic resonance spectroscopy to show that K1- and K2-modules are 3-cysteine-coordinated [Fe S ] clusters, with a histidine-derived nitrogen serving as the fourth ligand to K1 that is lost upon K1/K2-coupling. Further, we demonstrate that coexistence of SAM/K2-modules is a prerequisite for methyltransfer to K2 and hydrogen abstraction from the K2-associated methyl by a 5'-deoxyadenosyl radical. These results establish an important framework for mechanistic explorations of NifB while highlighting the utility of a synthetic-cluster-based reconstitution approach employed herein in functional analyses of iron-sulfur (FeS) enzymes.

Carbon monoxide dehydrogenase from Oligotropha carboxidovorans catalyzes the oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. In the oxidative half of the catalytic cycle, electrons gained from CO are passed intramolecularly through two [2Fe-2S] clusters and finally to a FAD cofactor. From FAD the electrons are ultimately passed to the electron transport chain of the Gram-negative organism. In the current study we have examined a variety of aspects of this enzyme in the oxidative- and reductive-half reactions and propose mechanisms for the oxidation of carbon monoxide and the proximal electron acceptor of the enzyme. First, we have identified the proximal acceptor of reducing equivalents. We have found CO dehydrogenase passes electrons directly to the quinone pool without using a cytochrome as an intermediary as had previously been proposed. This establishes a new category of redox-partner for the xanthine oxidase family of enzymes. Next, we examined the active site and find silver can be replaced for the active site copper. Cyanide effectively removes the copper and a Ag(I)-thiourea solution can reactive the enzyme, albeit at a lower turnover rate. The silver reconstitution can be verified by EPR, evident by the lack of coupling to the copper I=3/2 nucleus and in its place the sliver I=1/2 nucleus. This altered but active form of the protein is used to compare and contrast with the native copper-containing enzyme to develop a mechanism for CO oxidation. We then examined the EPR of CO dehydrogenase reduced by CO by electron nuclear double resonance spectroscopy (ENDOR). The ENDOR spectra of this state confirm that the 63,65Cu exhibits strong and almost entirely isotropic coupling, show that this coupling atypically has a positive sign, aiso = +148 MHz. When the intermediate is generated using 13CO, coupling to the 13C is observed, with a iso = +17.3 MHz. A comparison with the couplings seen in related, structurally assigned Mo(V) species from xanthine oxidase leads us to conclude that the intermediate contains a partially reduced, Mo(V)/Cu(I), center with CO bound at the copper. We next further characterized the kinetics and mechanisms of hydrogenase activity previously reported and find CO dehydrogenase effectively catalyzes H2 oxidation to protons. This activity is found to be independent of pH and does not appear to be reversible. A new EPR signal was found and is attributed to the H2 bound state with the molybdenum in an oxidation state, Mo(V), that prevents further catalysis. Finally, we have examined the inhibition of the enzyme by n-butylisonitrile and bicarbonate. We find that n-butylisonitrile reduces the and irreversibly inhibits the enzyme as is suggested by the crystal structure and computational studies previously reported. Bicarbonate acts as an uncompetitive inhibitor, reducing vmax and Km, while also producing a new EPR signal of the bicarbonate complex.