Reduction of N by nitrogenases occurs at an organometallic iron cofactor that commonly also contains either molybdenum or vanadium. The well-characterized resting state of the cofactor does not bind substrate, so its mode of action remains enigmatic. Carbon monoxide was recently found to replace a bridging sulfide, but the mechanistic relevance was unclear. Here we report the structural analysis of vanadium nitrogenase with a bound intermediate, interpreted as a μ-bridging, protonated nitrogen that implies the site and mode of substrate binding to the cofactor. Binding results in a flip of amino acid glutamine 176, which hydrogen-bonds the ligand and creates a holding position for the displaced sulfide. The intermediate likely represents state E or E of the Thorneley-Lowe model and provides clues to the remainder of the catalytic cycle.
Nitrogenases are
responsible for biological nitrogen fixation,
a crucial step in the biogeochemical nitrogen cycle. These enzymes
utilize a two-component protein system and a series of iron–sulfur
clusters to perform this reaction, culminating at the FeMco active
site (M = Mo, V, Fe), which is capable of binding and reducing N2 to 2NH3. In this review, we summarize how different
spectroscopic approaches have shed light on various aspects of these
enzymes, including their structure, mechanism, alternative reactivity,
and maturation. Synthetic model chemistry and theory have also played
significant roles in developing our present understanding of these
systems and are discussed in the context of their contributions to
interpreting the nature of nitrogenases. Despite years of significant
progress, there is still much to be learned from these enzymes through
spectroscopic means, and we highlight where further spectroscopic
investigations are needed.
Cofactors play key roles in metabolic pathways. Among them F(420) has proved to be a very attractive target for the selective inhibition of archaea and actinobacteria. Its biosynthesis, in a unique manner, involves a key enzyme, F(0)-synthase. This enzyme is a large monomer in actinobacteria, while it is constituted of two subunits in archaea and cyanobacteria. We report here the purification of both types of F(0)-synthase and their in vitro activities. Our study allows us to establish that F(0)-synthase, from both types, uses 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine as substrates but not 4-hydroxylphenylpyruvate as previously suggested. Furthermore, our data support the fact that F(0)-synthase generates two 5'-deoxyadenosyl radicals for catalysis which is unprecedented in reaction catalyzed by radical SAM enzymes.
The alternative, vanadium-dependent nitrogenase is employed by Azotobacter vinelandii for the fixation of atmospheric N under conditions of molybdenum starvation. While overall similar in architecture and functionality to the common Mo-nitrogenase, the V-dependent enzyme exhibits a series of unique features that on one hand are of high interest for biotechnological applications. As its catalytic properties differ from Mo-nitrogenase, it may on the other hand also provide invaluable clues regarding the molecular mechanism of biological nitrogen fixation that remains scarcely understood to date. Earlier studies on vanadium nitrogenase were almost exclusively based on a ΔnifHDK strain of A. vinelandii, later also in a version with a hexahistidine affinity tag on the enzyme. As structural analyses remained unsuccessful with such preparations we have developed protocols to isolate unmodified vanadium nitrogenase from molybdenum-depleted, actively nitrogen-fixing A. vinelandii wild-type cells. The procedure provides pure protein at high yields whose spectroscopic properties strongly resemble data presented earlier. Analytical size-exclusion chromatography shows this preparation to be a VnfDKG heterohexamer.
Coenzyme F420 is a redox cofactor found in methanogens
and in various actinobacteria. Despite the major biological importance
of this cofactor, the biosynthesis of its deazaflavin core (8-hydroxy-5-deazaflavin,
Fo) is still poorly understood. Fo synthase,
the enzyme involved, is an unusual multidomain radical SAM enzyme
that uses two separate 5′-deoxyadenosyl radicals to catalyze
Fo formation. In this paper, we report a detailed mechanistic
study on this complex enzyme that led us to identify (1) the hydrogen
atoms abstracted from the substrate by the two radical SAM domains,
(2) the second tyrosine-derived product, (3) the reaction product
of the CofH-catalyzed reaction, (4) the demonstration that this product
is a substrate for CofG, and (5) a stereochemical study that is consistent
with the formation of a p-hydroxybenzyl radical at
the CofH active site. These results enable us to propose a mechanism
for Fo synthase and uncover a new catalytic motif in radical
SAM enzymology involving the use of two 5′-deoxyadenosyl radicals
to mediate the formation of a complex heterocycle.
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