Spectroscopic and kinetic studies of Y114F and W116F mutants of Me2SO reductase from Rhodobacter capsulatus

Mutants of the active site residues Trp-116 and Tyr-114 of the molybdenum-containing Me2SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me2SO to reduced enzyme, the result of lower stability of the E-red center dot Me2SO complex. The absorption spectrum of this species ( but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wildtype enzyme, consistent with Tyr-114 interacting with bound Me2SO. The as-isolated W116F mutant is only five-coordinate, with one of the two equivalents of the pyranopterin cofactor found in the enzyme dissociated from the molybdenum and replaced by a second Mo=O group. Reduction of the mutant with sodium dithionite and reoxidation with Me2SO, however, regenerates the long-wavelength absorbance of functional enzyme, although the wavelength maximum is shifted to 670 nm from the 720 nm of wild-type enzyme. This "redox-cycled" mutant exhibits a Me2SO reducing activity and overall reaction mechanism similar to that of wild-type enzyme but rapidly reverts to the inactive five-coordinate form in the course of turnover