E.p.r. characterisation of the molybdenum centre of Rhodobacter capsulatus dimethylsulphoxide reductase: new signals on reduction with Na2S2O4.

Dimethylsulphoxide reductase (DMSOR) from the photosynthetic bacterium Rhalobacter capsulatus is a periplasmic respiratory oxomolybdenum enzyme which can utilise dimethylsulphoxide, trimethylamine N-oxide and chlorate as electron acceptors [1,2]. The enzyme, with the closely related one from R. sphaeroides [3], appears [4] to be unique in containing the molybdenum cofactor as the sole redox centre, and therefore the potential for optical study of the molybdenum centre is large. Preliminary e.p.r. studies [3,5,6] of DMSOR have demonstrated that the enzyme can display a t least two different MOW) e.p.r. signals. Dithiolene S+ MOW) charge transfer transitions have been identified [5,61 as contributing to magnetic circular dichroism spectra from DMSOR. However, detailed characterisation of the MOW) ion by e.p.r. has not previously been carried out. A number of MOW) e.p.r. signals were generated from DMSOR and are shown in Fig. 1. The close agreement between experimental spectra and computer simulations indicates that each signal corresponds to a single chemical species. The signals shown in (a) and (b) are comparable to those reported previously [3,5,61, though we estimated the e.p.r. parameters (data not shown) considerably more precisely than had been done in the earlier work, and we also studied the effects of buffer ions and of various additives, These two signals are quite like signals ['I] from E. coli nitrate reductase but, unlike those from that enzyme, are not in pH-dependent equilibrium with one another. On reduction of DMSOR under specific conditions with Na$,O,, we observed two new signals (Fig 1 c and d) a t considerably lower g-values. These are quite unlike the other signals from DMSOR. Both show splitting from interaction of Mo(V) with a single strongly coupled proton. Parameters for the signal of E'ig.l(c) were: g1,2,3 1.9702, 1.9678, 1.9560; A(1H)l,z,3 1.29, 1.15, 1.27 mT. These parameters for the signal of Fig.l(c) are similar to the parameters of the Slow signal from xanthine oxidase in the desulpho form and particularly to the Nitrate form of that signal [8]. This is the first instance of an enzyme exhibiting MOW) e.p.r. signals typical of more than one oxomolybdenum enzyme family kf.91 and suggests an unusual degree of flexibility of the molybdenum centre of DMSOR. Comparison with data on model compounds [e.g. 103 makes it clear that either a change of a ligand atom (e.g. from S to 0 or N) or a significant change of coordination geometry is required to convert the molybdenum centre of DMSOR from the states giving the signals of Figs l(a) and (b) to those giving the signals of Figs l(c) and (d). Since the latter signals were obtained