Towards the reaction mechanism of xanthine oxidase from EPR studies.

Introduction EPR was the first spectroscopic method other than UV/visible spectroscopy to be applied systematically to investigations of the structure and functioning of metalloenzymes. The first such EPR studies of xanthine oxidase were reported in 1959 [l]. Since one reduced state of each of the four redox-active centres in the molecule is EPRactive, there is obviously considerable potential for EPR work on the enzyme. This potential was enhanced by the early development [Z] of a fast kinetic EPR technique. It happens that the M o o oxidation state of molybdenum, in comparison with paramagnetic states of most transition metal ions, yields EPR signals that are particularly informative. This is because of the unusually narrow EPR linewidths to which M o o gives rise, supplemented by the particular mixture of magnetic and non-magnetic isotopes occurring in natural abundance in molybdenum. Thus, of all enzymes studied by EPR, xanthine oxidase has turned out (see, e.g. [3-51) to be the one most amenable to such investigations. In fact, supplemented by stopped-flow, E M S , electron nuclear double resonance (ENDOR) spectroscopy and more conventional data, EPR studies over what may appear a somewhat leisurely time span, had led to the main features of the xanthine oxidase reaction mechanism occurring at the molybdenum centre having been delineated [6-91 before the advent of information from X-ray crystallography [10,1 l]. This review will concentrate on two aspects of the reaction mechanism for which direct evidence has for some while been available in the literature from spectroscopic work. The first is the finding that the site to which the substrate proton displaced in the catalytic reaction is transferred is the sulphido ligand of molybdenum [5-71. This is now widely accepted in the literature. The second is the conclusion that transfer to the substrate molecule of the 0x0 ligand atom on molybdenum, widely advocated in relation to the mechanisms of enzymes such as DMSO reductase [12], does not occur with