No abstract

Molybednum-containing enzymes (Coughlan, 1980; Spiro, 1985) occupy a significant place in the development of the field now termed inorganic biochemistry. The importance of the metal as a biological trace element depends on its involvement in the known, and perhaps other as yet unknown, molybdoenzymes. That it plays a role in biological nitrogen fixation, the process whereby the enzyme nitrogenase in the root nodules of plants converts atmospheric nitrogen into ammonia, was recognized in the 1930s. The metal is also a constituent of a variety of other enzymes, having first been found in a mammalian enzyme, xanthine oxidase, in the 1950s.

Reactions of H(2)O(2) with superoxide dismutase were studied by e.p.r. (electron paramagnetic resonance) spectroscopy and other methods. In agreement with earlier work, the Cu(2+) of the enzyme is reduced by H(2)O(2), although the reaction does not go to completion and its kinetics are not simple. With dilute enzyme the time for half-reduction with 9mm-H(2)O(2) is about 150ms. It is suggested that the reaction is a one-electron reduction, involving liberation of O(2) (-). On somewhat more prolonged exposure to H(2)O(2), the enzyme is inactivated. For enzyme in dilute solution and over a limited range of H(2)O(2) concentrations, inactivation is first-order with respect to enzyme and reagent, with k=3.1m(-1).s(-1) at 20-25 degrees C. Inactivation is accompanied by marked changes in the e.p.r. and visible spectra and appears to be associated with destruction of one histidine residue per subunit. It is suggested that this histidine is close to the metal in the native enzyme and essential for its enzymic activity.

The dimethylsulphoxide reductase of Rhodobacter capsulatus contains a pterin molybdenum cofactor molecule as its only prosthetic group. Kinetic studies were consistent with re-oxidation of the enzyme being rate limiting in the turnover of dimethylsulphoxide in the presence of the benzyl viologen radical. EPR spectra of molybdenum(V) were generated by reducing the highly purified enzyme under a variety of conditions, and with careful control it was possible to generate at least five clearly distinct EPR signals. These could be simulated, indicating that each corresponds to a single chemical species. Structures of the signal-giving species are discussed in light of the EPR parameters and of information from the literature. Three of the signals show coupling of molybdenum to an exchangeable proton and, in the corresponding species, the metal is presumed to bear a hydroxyl ligand. One signal with g,, 1.96 shows a very strong similarity to a signal for the desulpho form of xanthine oxidase, while two others with g,, values of 1.98 show a distinct similarity to signals from nitrate reductase of Escherichia coli. These data indicate an unusual flexibility in the active site of dimethylsulphoxide reductase, as well as emphasising structural similarities between molybdenum enzymes bearing different forms of the pterin cofactor. Interchange among the different species must involve either a change of coordination geometry, a ligand exchange, or both. The latter may involve replacement of an amino acid residue co-ordinating molybdenum via 0 or N, for a cysteine co-ordinating via S. Since the two signals with g,, 1.96 were obtained only under specific conditions of reduction of the enzyme by dithionite, it is postulated that their generation may be triggered by reduction of the pteridine of the molybdenum cofactor from a dihydro state to the tetrahydro state.Enzymes that depend on the pterin molybdenum cofactor (Rajagopalan, 1991) are widely distributed, have a variety of important roles in different organisms and have been extensively studied in many laboratories (for recent reviews, see Spiro, 1985;Bray, 1988;Solomonson and Barber, 1990;Wootton et al., 1991 ;Enemark and Young, 1993). Detailed structural information from X-ray crystallography is not yet available on any of these enzymes (however, see Romao et al., 1993a,b). All but one of the enzymes contain, in addition to molybdenum, redox centres such as flavin, haem or ironsulphur. These complicate spectroscopic investigations and, in particular, their strong absorption in the ultraviolethisible region effectively masks the rather weak absorption of the molybdenum chromophore. Nevertheless, other spectroscopic methods have provided important insights into the local structure around the molybdenum atom. EPR spectroscopy has been very important in this context and has been supplemented particularly by extended X-ray absorption fine structure (EXAFS) and to a lesser extent by magnetic circular Abbreviations. MCD, magnetic circular dichroism ; EXAFS, extended X-ray absorpt...