Microcoulometric analysis of trimethylamine dehydrogenase.
Trimethylamine dehydrogenase, which contains one covalently bound 6-S-cysteinyl-FMN and one Fe4S4 cluster per subunit of molecular mass 83,000 Da, was purified to homogeneity from the methylotrophic bacterium W3A1. Microcoulometry at pH 7 in 50 mM-Mops buffer containing 0.1 mM-EDTA and 0.1 M-KCl revealed that the native enzyme required the addition of 3 reducing equivalents per subunit for complete reduction. In contrast, under identical conditions the phenylhydrazine-inhibited enzyme required the addition of 0.9 reducing equivalent per subunit with a midpoint potential of +110 mV. Least-squares analysis of the microcoulometric data obtained for the native enzyme, assuming uptake of 1 electron by Fe4S4 and 2 electrons by FMN, indicated midpoint potentials of +44 mV and +36 mV for the FMN/FMN.- and FMN.-/FMNH2 couples respectively and +102 mV for reduction of the Fe4S4 cluster.
Barber
M J
MJ
Department of Biochemistry and Molecular Biology, University of South Florida, College of Medicine, Tampa 33612.
Pollock
V
V
Spence
J T
JT
D005296:Ferrous Compounds; D005486:Flavin Mononucleotide; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010659:Phenylhydrazines / Q000494:pharmacology
D005296:Ferrous Compounds; D010659:Phenylhydrazines; C030299:phenylhydrazine; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; C022597:ferrous sulfide