Active site heterogeneity in dimethyl sulfoxide reductase from Rhodobactercapsulatus revealed by raman spectroscopy

Raman spectroscopy has been used to investigate the structure of the molybd enum cofactor in DMSO reductase from Rhodobacter capsulatus. Three oxidized forms of the enzyme, designated 'redox cycled', 'as prepared', and DMSORmo dD, have been studied using 752 nm laser excitation. In addition, two reduc ed forms of DMSO reductase, prepared either anaerobically using DMS or usin g dithionite, have been characterized. The 'redox cycled' form has a single band in the Mo=O stretching region at 865 cm(-1) consistent with other stu dies. This oxo ligand is found to be exchangeable directly with (DMSO)-O-18 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 evi dence from the labeling studies for a modified dithiolene sulfur atom, whic h could be present as a sulfoxide. In addition to the 865 cm(-1) band, an e xtra band at 818 cm(-1) is observed in the Mo=O stretching region of the 'a s 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 81 8 cm(-1) is assigned to the S=O stretch of a coordinated DMSO molecule. The DMSORmodD form, identified by its characteristic Raman spectrum, is also p resent in the 'as prepared' enzyme preparation but not after redox cycling. The complex mixture of forms identified in the 'as prepared' enzyme reveal s a substantial degree of active site heterogeneity in DMSO reductase.