X-ray absorption spectroscopy of dimethylsulfoxide reductase from Rhodobacter capsulatus

Abstract

 Mo K-edge X-ray absorption spectroscopy (XAS) has been used to probe the environment of Mo in dimethylsulfoxide (DMSO) reductase from Rhodobacter capsulatus in concert with protein crystallographic studies. The 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, the extended X-ray absorption fine structure (EXAFS) is also consistent with a seven-coordinate site. The site is composed of one oxo-group (Mo=O 1.71 Å), four S atoms (considered to arise from the dithiolene groups of the two molybdopterins, two at 2.32 Å and two at 2.47 Å, and two O atoms, one at 1.92 Å (considered to be H-bonded to Trp 116) and one at 2.27 Å (considered to arise from Ser 147). The Mo K-edge XAS recorded for single crystals of oxidised (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 enzyme 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 with the lack of a significant presence of Mo^V in the samples as monitored by EPR spectroscopy, are taken to indicate the presence of Mo^IV. For the dithionite-reduced sample, the Mo K-edge EXAFS indicates a coordination environment for Mo of two O atoms, one at 2.05 Å and one at 2.51 Å, and four S atoms at 2.36 Å. The coordination environment of the Mo in the DMS-reduced form of the enzyme involves three O atoms, one at 1.69 Å, one at 1.91 Å and one at 2.11 Å, plus four S atoms, two at 2.28 Å and two at 2.37 Å. The EXAFS and the protein crystallographic results for the 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 Å.