Affiliation:
1. Microbiology Group, Biology Department, Indiana University, Bloomington, Indiana 47405
Abstract
A soluble yellow CO dehydrogenase from CO-autotrophically grown cells of
Pseudomonas carboxydohydrogena
was purified 35-fold in seven steps to better than 95% homogeneity with a yield of 30%. The final specific activity was 180 μmol of acceptor reduced per min per mg of protein as determined by an assay based on the CO-dependent reduction of thionin. Methyl viologen, nicotinamide adenine dinucleotide (phosphate), flavin mononucleotide, and flavin adenine dinucleotide were not reduced by the enzyme, but methylene blue, thionin, and toluylene blue were reduced. The molecular weight of native enzyme was determined to be 4 × 10
5
. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed at least three nonidentical subunits of molecular weights 14,000 (α), 28,000 (β), and 85,000 (γ). The ratio of densities of each subunit after electrophoresis was about 1:2:6 (α/β/γ), suggesting an α
3
β
3
γ
3
structure for the enzyme. The purified enzyme was free of formate dehydrogenase and nicotinamide adenine dinucleotide-specific hydrogenase activities, but contained particulate hydrogenase-like activity with thionin as electron acceptor. Known metalchelating agents tested had no effect on CO dehydrogenase activity. No divalent cations tested stimulated enzyme activity. The native enzyme does not contain Ni since cells assimilated little
63
Ni during growth, and the specific
63
Ni content of the enzyme declined during purification. The isoelectric point of the native enzyme was found to be 4.5 to 4.7. The
K
m
for CO was found to be 63 μM. The spectrum of the enzyme and its protein-free extract revealed that it contains bound flavin. The cofactor was flavin adenine dinucleotide based on enzyme digestion and thin-layer chromatography. One mole of native enzyme contains at least 3 mol of noncovalently bound flavin adenine dinucleotide.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Cited by
65 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献