Active Site Heterogeneity in Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus Revealed by Raman Spectroscopy † Alasdair F. Bell, ‡ Xiang He, ‡ Justin P. Ridge, § Graeme R. Hanson, | Alastair G. McEwan, § and Peter J. Tonge* ,‡ Department of Chemistry, SUNY at Stony Brook, Stony Brook, New York 11794-3400, and School of Molecular & Microbial Sciences and Centre for Magnetic Resonance, UniVersity of Queensland, St. Lucia, Queensland 4072, Australia ReceiVed August 31, 2000; ReVised Manuscript ReceiVed NoVember 2, 2000 ABSTRACT: Raman spectroscopy has been used to investigate the structure of the molybdenum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated ‘redox cycled’, ‘as prepared’, and DMSOR mod D, have been studied using 752 nm laser excitation. In addition, two reduced forms of DMSO reductase, prepared either anaerobically using DMS or using dithionite, have been characterized. The ‘redox cycled’ form has a single band in the ModO stretching region at 865 cm -1 consistent with other studies. This oxo ligand is found to be exchangeable directly with DMS 18 O or by redox cycling. Furthermore, deuteration experiments demonstrate that the oxo ligand in the oxidized enzyme has some hydroxo character, which is ascribed to a hydrogen bonding interaction with Trp 116. There is also evidence from the labeling studies for a modified dithiolene sulfur atom, which could be present as a sulfoxide. In addition to the 865 cm -1 band, an extra band at 818 cm -1 is observed in the ModO stretching region of the ‘as prepared’ enzyme which is not present in the ‘redox cycled’ enzyme. Based on the spectra of unlabeled and labeled DMS reduced enzyme, the band at 818 cm -1 is assigned to the SdO stretch of a coordinated DMSO molecule. The DMSOR mod D form, identified by its characteristic Raman spectrum, is also present in the ‘as prepared’ enzyme preparation but not after redox cycling. The complex mixture of forms identified in the ‘as prepared’ enzyme reveals a substantial degree of active site heterogeneity in DMSO reductase. The mononuclear molybdoenzymes form a diverse but coherent group, characterized by the presence of one or two molecules of molybdopterin with dithiolene groups coordi- nated to a molybdenum center (1-3). Almost all molyb- doenzymes catalyze a two-electron-transfer reaction that is linked to oxygen atom transfer to or from a water molecule. During catalysis, the molybdenum cycles between the Mo- (VI) and Mo(IV) states. Dimethyl sulfoxide (DMSO) reduc- tase from photosynthetic bacteria of the genus Rhodobacter is regarded as a key molybdenum enzyme for studies of structure-function relationships since it is one of only a very few examples of an enzyme which contains a molybdenum cofactor as its only prosthetic group. Thus, spectroscopic studies can proceed without interference from other chro- mophoric groups. The reaction catalyzed by this enzyme is shown in Figure 1. Our understanding of the structure of the molybdenum site in this enzyme has been transformed by X-ray crystallography (4-8), and this has been comple- mented by analysis using X-ray absorption spectroscopy (XAS) (9-11), resonance Raman spectroscopy (12-14), and EPR spectroscopy (15). X-ray crystallographic studies have produced a surprisingly complex picture of the active site in DMSO reductase as three distinct, independent cofactor structures have been proposed (4-6). These structures differ from one another in two important respects: the number of oxo ligands and the nature of coordination of the dithiolene ligands. They do, however, agree in the overall structure of the protein and on the direct coordination of the cofactor to the protein via serine-147. A number of EXAFS studies have appeared which have helped to clarify some aspects of the structure of the molybdenum cofactor. In particular, the most recent EXAFS study on recombinant DMSO reductase from Rhodobacter sphaeroides proposed a six-coordinate cofactor structure which has a single oxo ligand and two equivalent dithiolene ligands and is also coordinated to serine-147 (11). A consensus has emerged that this oxo group (O2) is the one which hydrogen bonds to W116 since this is the oxo group which reacts with DMS to form a species with DMSO bound at the active site (7). This view is reinforced by the position of the single oxo in the revised crystal structure of DMSO reductase from R. sphaeroides (8). However, as a result, the oxo group that hydrogen bonds to Y114 (known as O1 in the structures described by Bailey and co-workers), and identified as the only oxo group in the first crystal † This work was supported by NSF Grant MCB9604254, by US Army Research Office Grant DAAG559710083, and by NIH Grant AI44639 to P.J.T., by a grant from the Australian Research Council to A.G.M. and G.R.H., and by a postdoctoral fellowship to A.F.B. from the American Heart Association. * To whom correspondence should be addressed. Telephone: (631) 632 7907, Fax: (631) 632 7960, Email: Peter.Tonge@sunysb.edu. ‡ Department of Chemistry, SUNY at Stony Brook. § School of Molecular & Microbial Sciences, University of Queen- sland. | Centre for Magnetic Resonance, University of Queensland. FIGURE 1: Reaction catalyzed by DMSO reductase. 440 Biochemistry 2001, 40, 440-448 10.1021/bi002065k CCC: $20.00 © 2001 American Chemical Society Published on Web 12/15/2000