The High Resolution Crystal Structure of DMSO Reductase in Complex with DMSO A. S. McAlpine 1 , A. G. McEwan 2 and S. Bailey 1 * 1 CCLRC Daresbury Laboratory Daresbury, Warrington Cheshire, WA4 4AD, UK 2 Department of Microbiology University of Queensland Brisbane, Queensland Australia The crystal structure of the molybdenum enzyme dimethylsulphoxide reductase (DMSOR) has been determined at 1.9 A Ê resolution with sub- strate bound at the active site. DMSOR is an oxotransferase which cata- lyses the reduction of dimethylsulphoxide (DMSO) to dimethylsulphide (DMS) in a two stage reaction which is linked to oxygen atom transfer and electron transfer. In the ®rst step, DMSO binds to reduced (Mo (IV) ) enzyme, the enzyme is oxidised to Mo (VI) with an extra oxygen ligand and DMS is released. Regeneration of reduced enzyme is achieved by transfer of two electrons, successively from a speci®c cytochrome, and release of the oxygen as water. The enzyme, puri®ed under aerobic con- ditions, is in the oxidised (Mo (VI) ) state. Addition of a large excess of DMS to the oxidised enzyme in solution causes a change in the absorp- tion spectrum of the enzyme. The same reaction occurs within crystals of the enzyme and the crystal structure reveals a complex with DMSO bound to the molybdenum via its oxygen atom. X-ray edge data indicate that the metal is in the Mo (IV) state. The DMSO ligand replaces one of the two oxo groups which ligate the oxidised form of the enzyme, suggesting very strongly that this is the oxygen which is transferred during catalysis. Residues 384 to 390, disordered in the oxidised enzyme, are now clearly seen in the cleft leading to the active site. The side-chain of Trp388 forms a lid trapping the substrate/product. # 1998 Academic Press Limited Keywords: dimethylsulphoxide reductase; dimethyl sulphide; molybdenum; crystal structure; molybdopterin *Corresponding author Introduction Molybdenum is present at the active site of many enzymes important in nitrogen and sulphur metabolism (Stiefel, 1993). With the exception of nitrogenase, molybdenum is present in proteins in the form of a molybdopterin cofactor (Moco; Rajagopalan, 1991). All forms of Moco are based on an organic component known as molybdopterin which consists of a modi®ed pterin ring with an ene-dithiolate (dithiolene) side-chain, responsible for ligating the molybdenum. In many bacterial oxomolybdenum enzymes the molybdopterin is in the form of a dinucleotide species and in dimethyl- sulphoxide reductase (DMSOR) it is established that it is molybdopterin guanine dinucleotide (MGD; Johnson et al., 1991). Recently, it has been found that there are two MGD units per molyb- denum (Hilton & Rajagopalan, 1996). DMSOR catalyses the reduction of di- methylsulphoxide (DMSO) in the reaction: Me 2 SO 2e   2H  () Me 2 S  H 2 O. The reaction is a two stage process. In the ®rst step, the reduced form (Mo (IV) ) of the enzyme binds DMSO and dimethylsulphide (DMS) is released, leaving the enzyme in its oxidised state with the oxygen co- ordinated to Mo (VI) . In the second step, two elec- trons are transferred to the molybdenum from a speci®c cytochrome and the oxygen is protonated to form water. The molybdenum at the active site of the enzyme thus cycles between the Mo (IV) and Mo (VI) states, possibly via a Mo (V) intermediate in the second step, as electron transfer from the cyto- chrome is by single electron steps. Evidence for the direct transfer of the DMSO oxygen has come from 18 O labelling experiments (Schultz et al., 1995). Experiments with chemical systems, designed to mimic oxygen transfer at the molybdenum active site, have suggested a di-oxo Mo (VI) centre in the Abbreviations used: Moco, molybdopterin cofactor; DMSO, dimethylsulphoxide; DMSOR, dimethylsulphoxide reductase; MGD, molybdopterin guanine dinucleotide; XAS, X-ray absorption spectroscopy; DCIP, 2,6, dichloroindophenol; PES, phenazine ethosulphate. J. Mol. Biol. (1998) 275, 613±623 0022±2836/98/040613±11 $25.00/0/mb971513 # 1998 Academic Press Limited