Identity and function of an essential nitrogen ligand of the nitrogenase cofactor biosynthesis protein NifB.

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 [Fe4S4] clusters (designated K1 and K2) concomitant with carbide insertion into an [Fe8S9C] 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 (MaNifB) as the nitrogen ligand for K1. Biochemical and continuous wave electron paramagnetic resonance data demonstrate the inability of MaNifB 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 MaNifB 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

Dioxygen Activation by Nonheme Diiron Enzymes: Diverse Dioxygen Adducts, High-Valent Intermediates, and Related Model Complexes

A growing subset of metalloenzymes activates dioxygen with nonheme diiron active sites to effect substrate oxidations that range from the hydroxylation of methane and the desaturation of fatty acids to the deformylation of fatty aldehydes to produce alkanes and the six-electron oxidation of aminoarenes to nitroarenes in the biosynthesis of antibiotics. A common feature of their reaction mechanisms is the formation of O₂ adducts that evolve into more reactive derivatives such as diiron­(II,III)-superoxo, diiron­(III)-peroxo, diiron­(III,IV)-oxo, and diiron­(IV)-oxo species, which carry out particular substrate oxidation tasks. In this review, we survey the various enzymes belonging to this unique subset and the mechanisms by which substrate oxidation is carried out. We examine the nature of the reactive intermediates, as revealed by X-ray crystallography and the application of various spectroscopic methods and their associated reactivity. We also discuss the structural and electronic properties of the model complexes that have been found to mimic salient aspects of these enzyme active sites. Much has been learned in the past 25 years, but key questions remain to be answered

The Fe Protein: An Unsung Hero of Nitrogenase

Although the nitrogen-fixing enzyme nitrogenase critically requires both a reductase component (Fe protein) and a catalytic component, considerably more work has focused on the latter species. Properties of the catalytic component, which contains two highly complex metallocofactors and catalyzes the reduction of N2 into ammonia, understandably making it the “star” of nitrogenase. However, as its obligate redox partner, the Fe protein is a workhorse with multiple supporting roles in both cofactor maturation and catalysis. In particular, the nitrogenase Fe protein utilizes nucleotide binding and hydrolysis in concert with electron transfer to accomplish several tasks of critical importance. Aside from the ATP-coupled transfer of electrons to the catalytic component during substrate reduction, the Fe protein also functions in a maturase and insertase capacity to facilitate the biosynthesis of the two-catalytic component metallocofactors: fusion of the [Fe8S7] P-cluster and insertion of Mo and homocitrate to form the matured [(homocitrate)MoFe7S9C] M-cluster. These and key structural-functional relationships of the indispensable Fe protein and its complex with the catalytic component will be covered in this review

Reactivity, Mechanism, and Assembly of the Alternative Nitrogenases

Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which facilitates the cleavage of the relatively inert triple bond of N2. Nitrogenase is most commonly associated with the molybdenum-iron cofactor called FeMoco or the M-cluster, and it has been the subject of extensive structural and spectroscopic characterization over the past 60 years. In the late 1980s and early 1990s, two "alternative nitrogenase" systems were discovered, isolated, and found to incorporate V or Fe in place of Mo. These systems are regulated by separate gene clusters; however, there is a high degree of structural and functional similarity between each nitrogenase. Limited studies with the V- and Fe-nitrogenases initially demonstrated that these enzymes were analogously active as the Mo-nitrogenase, but more recent investigations have found capabilities that are unique to the alternative systems. In this review, we will discuss the reactivity, biosynthetic, and mechanistic proposals for the alternative nitrogenases as well as their electronic and structural properties in comparison to the well-characterized Mo-dependent system. Studies over the past 10 years have been particularly fruitful, though key aspects about V- and Fe-nitrogenases remain unexplored

Structural evidence for a dynamic metallocofactor during N2 reduction by Mo-nitrogenase

The enzyme nitrogenase uses a suite of complex metallocofactors to reduce dinitrogen (N2) to ammonia. Mechanistic details of this reaction remain sparse. We report a 1.83-angstrom crystal structure of the nitrogenase molybdenum-iron (MoFe) protein captured under physiological N2 turnover conditions. This structure reveals asymmetric displacements of the cofactor belt sulfurs (S2B or S3A and S5A) with distinct dinitrogen species in the two αβ dimers of the protein. The sulfur-displaced sites are distinct in the ability of protein ligands to donate protons to the bound dinitrogen species, as well as the elongation of either the Mo-O5 (carboxyl) or Mo-O7 (hydroxyl) distance that switches the Mo-homocitrate ligation from bidentate to monodentate. These results highlight the dynamic nature of the cofactor during catalysis and provide evidence for participation of all belt-sulfur sites in this process

A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β‑Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase

The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor l-<i>p</i>-aminophenylalanine (l-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster that is reduced to the diferrous state (<b>WT</b>^<b>R</b>) to initiate O₂ activation. However, rapid O₂ activation occurs only when <b>WT</b>^<b>R</b> is bound to CmlP, the NRPS to which l-PAPA is covalently attached. Here the X-ray crystal structure of <b>WT</b>^<b>R</b> is reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the <b>WT</b>^<b>R</b> cluster ligand structure as well as the structures of <b>WT</b>^<b>R</b> in complex with a functional CmlP variant (CmlP_AT) with and without l-PAPA attached. It is found that formation of the active <b>WT</b>^<b>R</b>:CmlP_AT–l-PAPA complex converts at least one iron of the cluster from six- to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of <b>WT</b>^<b>R</b> is E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O₂ in the presence or absence of CmlP_AT–l-PAPA, showing loss of regulation. However, this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O₂ reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases

Unprecedented (μ-1,1-Peroxo)diferric Structure for the Ambiphilic Orange Peroxo Intermediate of the Nonheme <i>N</i>‑Oxygenase CmlI

The final step in the biosynthesis of the antibiotic chloramphenicol is the oxidation of an aryl-amine substrate to an aryl-nitro product catalyzed by the <i>N</i>-oxygenase CmlI in three two-electron steps. The CmlI active site contains a diiron cluster ligated by three histidine and four glutamate residues and activates dioxygen to perform its role in the biosynthetic pathway. It was previously shown that the active oxidant used by CmlI to facilitate this chemistry is a peroxo-diferric intermediate (<b>CmlI</b>^<b>P</b>). Spectroscopic characterization demonstrated that the peroxo binding geometry of <b>CmlI</b>^<b>P</b> is not consistent with the μ-1,2 mode commonly observed in nonheme diiron systems. Its geometry was tentatively assigned as μ–η²:η¹ based on comparison with resonance Raman (rR) features of mixed-metal model complexes in the absence of appropriate diiron models. Here, X-ray absorption spectroscopy (XAS) and rR studies have been used to establish a refined structure for the diferric cluster of <b>CmlI</b>^<b>P</b>. The rR experiments carried out with isotopically labeled water identified the symmetric and asymmetric vibrations of an Fe–O–Fe unit in the active site at 485 and 780 cm^–1, respectively, which was confirmed by the 1.83 Å Fe–O bond observed by XAS. In addition, a unique Fe···O scatterer at 2.82 Å observed from XAS analysis is assigned as arising from the distal O atom of a μ-1,1-peroxo ligand that is bound symmetrically between the irons. The (μ-oxo)­(μ-1,1-peroxo)­diferric core structure associated with <b>CmlI</b>^<b>P</b> is unprecedented among diiron cluster-containing enzymes and corresponding biomimetic complexes. Importantly, it allows the peroxo-diferric intermediate to be ambiphilic, acting as an electrophilic oxidant in the initial <i>N-</i>hydroxylation of an arylamine and then becoming a nucleophilic oxidant in the final oxidation of an aryl-nitroso intermediate to the aryl-nitro product

Evidence of substrate binding and product release via belt-sulfur mobilization of the nitrogenase cofactor

The Mo-nitrogenase catalyses the ambient reduction of N2 to NH3 at the M-cluster, a complex cofactor that comprises two metal-sulphur partial cubanes ligated by an interstitial carbide and three belt-sulphurs. A recent crystallographic study suggests binding of N2 via displacement of the belt-sulphur(s) of the M-cluster upon turnover. However, the direct proof of N2 binding and belt-sulphur mobilization during catalysis remains elusive. Here we show that N2 is captured on the M-cluster via electron- and sulphur-depletion, and that the N2-captured state is catalytically competent in generating NH3. Moreover, we demonstrate that product release only occurs when sulphite is supplied along with a reductant, that sulphite is inserted as sulphide into the belt-sulphur displaced positions, and that there is a dynamic in-and-out of the belt-sulphurs during catalysis. Together, these results establish the mobilization of the cofactor belt-sulphurs as a crucial, yet overlooked, mechanistic element of the nitrogenase reaction

Spectroscopic Characterization of an Eight‐Iron Nitrogenase Cofactor Precursor that Lacks the “9 th

Nitrogenases catalyze the reduction of N2 to NH4+ at its cofactor site. Designated the M-cluster, this [MoFe7 S9 C(R-homocitrate)] cofactor is synthesized via the transformation of a [Fe4 S4 ] cluster pair into an [Fe8 S9 C] precursor (designated the L-cluster) prior to insertion of Mo and homocitrate. We report the characterization of an eight-iron cofactor precursor (designated the L*-cluster), which is proposed to have the composition [Fe8 S8 C] and lack the "9th sulfur" in the belt region of the L-cluster. Our X-ray absorption and electron spin echo envelope modulation (ESEEM) analyses strongly suggest that the L*-cluster represents a structural homologue to the l-cluster except for the missing belt sulfur. The absence of a belt sulfur from the L*-cluster may prove beneficial for labeling the catalytically important belt region, which could in turn facilitate investigations into the reaction mechanism of nitrogenases