X-RAY-ABSORPTION SPECTROSCOPY OF DIMETHYLSULFOXIDE REDUCTASE FROM RHODOBACTER-CAPSULATUS

Mo It-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodo bacter capsulatus in concert with protein crystallographic studies, Th e oxidised (Mo-VI) protein has been investigated in solution at 77 K, the Mo K-edge position (20006.4 eV) is consistent with the presence of Mo-VI and, in agreement with the protein crystallographic results, th e extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one ore-group (M o=O 1.71 Angstrom). four S atoms (considered to arise from the dithiol ene groups of the two molybdopterins, two at 2.32 Angstrom and two at 2.47 Angstrom, and two O atoms, one at 1.92 Angstrom (considered to be H-bonded to Trp 116) and one at 2.27 Angstrom (considered to arise fr om Ser 147). The Mo it-edge XAS recorded for single crystals of oxidis ed (Mo-VI) DMSO reductase at 77 K showed a close correspondence to the data for the frozen solution but had an inferior signal:noise ratio. The dithionite-reduced form of the enzyme and a unique form of the enz yme produced by the addition of dimethylsulfide (DMS) to the oxidised (Mo-VI) enzyme have essentially identical energies for the Mo K-edge, at 20004.4 eV and 20004.5 eV, respectively; these values, together wit h the lack of a significant presence of Mo-V in the samples as monitor ed by EPR spectroscopy, are taken to indicate the presence of Mo-IV, F or the dithionite-reduced sample, the Mo It-edge EXAFS indicates a coo rdination environment for Mo of two O atoms, one at 2.05 Angstrom and one at 2.51 Angstrom, and four S atoms at 2.36 Angstrom. The coordinat ion environment of the Mo in the DMS-reducod form of the enzyme involv es three O atoms, one at 1.69 Angstrom, one at 1.91 Angstrom and one a t 2.11 Angstrom, plus four S atoms, two at 2.28 Angstrom and two at 2. 37 Angstrom. The EXAFS and the protein crystallographic results for th e DMS-reduced form of the enzyme are consistent with the formation of the substrate, DMSO, bound to Mo-IV with an Mo-O bond of length 1.92 A ngstrom.