Molybdenum cofactor reduced form

The most efficient system of this type is obtained by the reduction of bovine serum albumin in the presence of molybdate. Apparently disulfide links in the peptide are broken and form thiolate groups which then bind molybdenum. In a borate buffer, this system will reduce dinitrogen and acetylene, although not using dithionite as an electron source. The turnover is similar to that of the iron-molybdenum cofactor (see Section XII), and dinitrogen reduction is inhibited by carbon monoxide and stimulated by ATP. The yield of ammonia is linearly dependent upon PN2, and the yield is also depressed in the presence of fumarate and, more surprisingly, succinate. It is calculated that the... 

In summary, a 6-substituted pterin was first identified as a structural component of the molybdenum cofactor from sulfite oxidase, xanthine oxidase and nitrate reductase in 1980 (24). Subsequent studies provided good evidence that these enzymes possessed the same unstable molyb-dopterin (1), and it seemed likely that 1 was a constituent of all of the enzymes of Table I. It now appears that there is a family of closely related 6-substituted pterins that may differ in the oxidation state of the pterin ring, the stereochemistry of the dihydropterin ring, the tautomeric form of the side chain, and the presence and nature of a dinucleotide in the side chain. In some ways the variations that are being discovered for the pterin units of molybdenum enzymes are beginning to parallel the known complexity of naturally occurring porphyrins, which may have several possible side chains, various isomers of such side chains, and a partially reduced porphyrin skeleton (46). 

These clusters are each composed of eight iron atoms and seven sulfide ions. In the reduced form, each cluster takes the form of two 4Fe-3S partial cubes linked by a central sulfide ion. Each cluster is linked to the protein through six cysteinate residues. Electrons flow from the P cluster to the FeMo cofactor, a very unusual redox center. Because molybdenum is present in this cluster, the nitrogenase component is also called the molybdenum-iron protein (MoFe protein). The FeMo cofactor consists of two M-3Fe-3S clusters, in which molybdenum occupies the M site in one cluster and iron occupies it in the other. The two clusters are joined by three sulfide ions. The FeMo cofactor is also coordinated to a homocitrate moiety and to the a subunit through one histidine residue and one cysteinate residue. This cofactor is distinct from the molybdenum-containing cofactor found in sulfite oxidase and apparently all other molybdenum-containing enzymes except nitrogenase. 

The Mo enzyme consists of two components (1) the Fe protein (molecular weight 57,000 daltons), which contains iron and sulfur (4 atoms of each per protein) and (2) the MoFe protein (220,000 daltons, Uz subunits), which contains both metals (1 atom Mo, 32 atoms Fe). Each also contains S ions (ca. one per iron), which act as bridging ligands for the metals. The protein contains special Fe—S clusters called P clusters that have EPR resonances like those of no other Fe—S cluster. A soluble protein-fiee molybdenum and iron-containing cluster can be separated from the enzyme. This iron-molybdenum cofactor, or FeMo-co, was known to have approximately 1 Mo, 7-8 Fe, 4-6 S ", and one molecule of homocitrate ion. As for the P cluster, there was no agreement on the structure of FeMo-co for many years. In purified form FeMo-co does crystallize, and it can restore N2 reducing activity to samples of mutant N2ase that are inactive because they lack FeMo-co. On the other hand, no crystal structure of FeMo-co proved possible, and no synthetic model complex was found that could activate the mutant enzyme. 

The molyhdopterin cofactor, as found in different enzymes, may be present either as the nucleoside monophosphate or in the dinucleotide form. In some cases the molybdenum atom binds one single cofactor molecule, while in others, two pterin cofactors coordinate the metal. Molyhdopterin cytosine dinucleotide (MCD) is found in AORs from sulfate reducers, and molyhdopterin adenine dinucleotide and molyb-dopterin hypoxanthine dinucleotide were reported for other enzymes (205). The first structural evidence for binding of the dithiolene group of the pterin tricyclic system to molybdenum was shown for the AOR from Pyrococcus furiosus and D. gigas (199). In the latter, one molyb-dopterin cytosine dinucleotide (MCD) is used for molybdenum ligation. Two molecules of MGD are present in the formate dehydrogenase and nitrate reductase. 

The detection of multiple forms of oxidized and semi-reduced FeMoco has important implications with regard to the structure and function of cofactor. Derivation of these species from the iron-molybdenum center in the protein is illustrated in Scheme 1. This behavior is based on the electrochemical and EPR results presented in the previous sections. 

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