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 Me(2)SO reductase from Rhodobacter capsulatus have been examined spectroscopically and kinetically. The Y114F mutant has an increased rate constant for oxygen atom transfer from Me(2)SO to reduced enzyme, the result of lower stability of the E(red).Me(2)SO complex. The absorption spectrum of this species (but not that of either oxidized or reduced enzyme) is significantly perturbed in the mutant relative to wild-type enzyme, consistent with Tyr-114 interacting with bound Me(2)SO. 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 Me(2)SO, 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 Me(2)SO 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.
Cobb
Nathan
N
Department of Molecular and Cellular Biochemistry, The Ohio State University, Columbus, Ohio 43210, USA.
Hemann
Craig
C
Polsinelli
Gregory A
GA
Ridge
Justin P
JP
McEwan
Alastair G
AG
Hille
Russ
R
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D004227:Dithionite / Q000737:chemistry; D007700:Kinetics; D008982:Molybdenum / Q000737:chemistry; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D016354:Rhodobacter capsulatus / Q000201:enzymology* / Q000235:genetics; D013053:Spectrophotometry
D001426:Bacterial Proteins; D004227:Dithionite; D008982:Molybdenum; D010088:Oxidoreductases; C100768:dimethylsulfide - acceptor oxidoreductase