Nitrogenase molybdenum enzymes

Not surprisingly, only about 20 of the chemical elements found on Earth are used by living organisms (Chapters 3 and 8). Most of them are common elements. Rare elements are used, if at all, only at extremely low concentrations for specialized functions. An example of the latter is the use of molybdenum as an essential component of nitrogenase, the enzyme that catalyzes the fixation of elemental dinitrogen. Because they are composed of common elements, living organisms exert their most profound effects on the cycles of those elements. 

In contrast with the molybdenum enzyme, hydrazine (N2H4) is a minor product from the reduction of N2 by vanadium nitrogenase (186). The production of N2H4 increases with increasing temperature up to 50°C, at which temperature the ability of vanadium nitrogenase to produce to NH3 ceases, although the production of N2H4 increases... 

The enzymes that utilize molybdenum can be grouped into two broad categories (1) the nitrogenases, where Mo is part of a multinu-clear metal center, or (2) the mononuclear molybdenum enzymes, such as xanthine oxidase (XO), dimethyl sulfoxide (DMSO) reductase, formate dehydrogenase (FDH), and sulfite oxidase (SO). The last... 

So little is known about molybdenum enzymes other than milk xanthine oxidase that there is little to be said by way of general conclusions. In all cases where there is direct evidence (except possibly for xanthine dehydrogenase from Micrococcus lactilyticus) it seems that molybdenum in the enzymes does have a redox function in catalysis. For the xanthine oxidases and dehydrogenases and for aldehyde oxidase, the metal is concerned in interaction of the enzymes with reducing substrates. However, for nitrate reductase it is apparently in interaction with the oxidizing substrate that the metal is involved. In nitrogenase the role of molybdenum is still quite uncertain. 

Recently, Brill and co-workers (43, 44) have isolated mutant strains of Azotobacter vinelandii which produce an inactive nitrogenase component. This component can be reactivated by treatment with the neutralized acid-hydrolysis products of other nitrogenases (which themselves become inactive on such a treatment) but not apparently with any other molybdenum enzymes. This may either reflect a difference between the cofactor in nitrogenase and other molybdenum enzymes or may be caused by the reconstitution conditions used which may not have been sufficiently varied to allow for different molybdenum oxidation states to be attained. In any event, the chemical characterization and authentication of the molybdenum cofactor should reveal some of the intimate details of the molybdenum site. 

The substrate half-reactions are displayed in Tables I and II. In each case, a two-electron process seems to be involved. Only in nitro-genase are greater numbers of electrons transferred, and the discussion earlier in this paper summarizes the evidence that these processes occur in two-electron steps. The two-electron reaction of the molybdenum site never appears to be simply an electron transfer reaction. In the case of nitrogenase, each substrate takes up an equal (or greater) number of protons to form the product. In the other molybdenum enzymes, proton transfer and addition or removal of H20 are also required. In each case, however, there is at least one proton transferred in the same direction as the pair of electrons. These data, taken in conjunction with the EPR evidence for proton transfer from the substrate to the active site in xanthine oxidase, suggest that the molybdenum site in all the enzymes... 

Mechanistic speculations about the molybdoenzymes must be considered to be in their infancy with the possible exception of those for xanthine oxidase. Although the detailed structural nature of the molybdenum site is unknown, there is sufficient information from biochemical and coordination chemistry studies to allow informed arguments to be drawn. Here we first discuss evidence for the nuclearity of the molybdenum site and then discuss both oxo-transfer and proton-electron transfer mechanisms for molybdenum enzymes. A final discussion considers the unique aspects of nitrogenase and the possible reasons for the use of molybdenum in enzymes. 

There are >40 distinct molybdenum enzymes that occur in all classes of living systems and are especially important in the biochemical cycles of carbon, nitrogen, and sulfur (24b). The majority of the molybdenum enzymes, with notable exceptions including the nitrogenases (25-28) and a 2-hydroxyglutaryl-CoA dehydratase (10), catalyze a conversion of the type [Eq. 1], that is, the net effect of the catalysis corresponds to the transfer of an oxygen atom to or from the substrate. 

Bolen, J.T., Cambasso, N., Muchmore, S.W., Morgan, T.V., and Mortenson, L. E. (1993) Structure and Environment of metal clusters of the nitrogenase molybdenum iron protein from Clostridium pasterianum, in Stiefel, E.I., Coucouvanis, D., and ewton, W.E. (eds.), Molibdenum Enzymes, Cofactors, and Model Systems, Am. Chem. Soc., Wahington, DC. 

EXAFS studies indicate very little change in the coordination of the Mo atom in Mo-nitrogenase during enzyme turnover an indication that N2 probably does not bind to molybdenum but instead to iron. However, this issue has not been completely resolved. 

R. H. Holm and E. D. Simhon, Molybdenum-tungsten-iron-sulfur Chemistry Current Status and Relevance to the Native Cluster of Nitrogenase, in Molybdenum Enzymes , ed. T. G. Spho, Wiley, New York, 1985, p. 1. 

The Fe protein of the vanadium nitrogenase, encoded by vnfH, shares a high degree of homology with the niJH-encoded Fe protein of the molybdenum nitrogenase. Like its counterpart in the molybdenum enzyme, the Fe protein of the vanadium nitrogenase has an U2 homodimeric composition and contains an [4Fe-4S] cluster. 

W. E. Newton and D. R. Dean, Role of the iron-molybdenum cofactor polypeptide environment in azotobacter vinelandii-nitrogenase catalysis, in Molybdenum Enzymes, Cofactors and Model Systems , eds. E. I. Stiefel, D. Coucouvanis, and... 

B. K. Burgess, Nitrogenase structure, function and genetics, in Molybdenum Enzymes, Cofactors and Model Systems , eds. 

Orme-Johnson, W.H., Miinck, E. On the Prosthetic Groups of Nitrogenase. In Molybdenum and Molybdenum Enzymes (ed. Coughlan, M.), Oxford, Pergamon Press 1980, pp. 429-438... 

---