ACTIVE-SITE STRUCTURES AND CATALYTIC MECHANISM OF RHODOBACTER-SPHAEROIDES DIMETHYL-SULFOXIDE REDUCTASE AS REVEALED BY RESONANCE RAMAN-SPECTROSCOPY

Resonance Raman spectra and excitation profiles (413-676 nm) are repor ted for four distinct forms of Rhodobacter sphaeroides dimethyl sulfox ide (DMSO) reductase: as prepared Mo(VI), dithionite-reduced Mo(IV), d imethyl sulfide reduced Mo(IV), and glycerol-inhibited Mo(V). All of t he vibrational modes in the 200-1700 cm(-1) region of the Mo(VI) and M o(IV) forms are assigned to vibrations involving atoms in the first or second coordination sphere of the bis-molybdopterin-coordinated Mo ac tive site, the dithiolene chelate rings, or nonresonantly enhanced pro tein modes. On the basis of O-18/O-16 isotope shifts, the Mo(VI) form is shown to be mono-ore with nu(Mo=O) at 862 cm(-1), and the DMS-reduc ed Mo(IV) form is shown to involve bound DMSO with nu(Mo-O) at 497 cm( -1) and nu(S=O) at 862 cm(-1). Bands at 536 and 513 cm(-1) are tentati vely assigned to nu(Mo-O(Ser)) stretching modes of coordinated serinat e in the Mo(VI) and Mo(IV) forms, respectively. The vibrational modes of two distinct types of dithiolene chelate rings are identified on th e basis of their excitation profiles, and the frequencies indicate tha t one is best viewed as a dithiolate ligand, while the other has more pi-delocalized character. In the low-frequency region between 335 and 405 cm(-1), the Mo-S stretching modes of a distorted square pyramidal MoS4 unit are assigned in each of the four derivatives investigated, b ased on the S-34 isotope shifts and sensitivity to Mo oxidation state. The average Mo-S bond strength increases with decreasing Mo oxidation state. Taken together, the Mo-S and dithiolene vibrational assignment s indicate that all four of the molybdopterin dithiolene S atoms remai n coordinated in each of the four forms investigated. Structures for e ach of these four derivatives are proposed on the basis of the resonan ce Raman results, and the ability to monitor directly the origin and f ate of the Mo oxo group via isotopic labeling indicates that each corr esponds to a catalytically competent intermediate in the reaction cycl e. Overall, the results provide direct confirmation of an oxygen atom transfer mechanism, with the active site cycling between mono-oxo-Mo(V I) and des-oxo-Mo(IV) forms via a DMSO-bound Mo(IV) intermediate, and the molybdopterin dithiolene ligands staying firmly attached throughou t the catalytic cycle.