Characterization of NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system from Pseudomonas arvilla c-1.

NADH-cytochrome c reductase, a component of benzoate 1,2-dioxygenase system, was purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis and ultracentrifugation, from benzoate-induced cells of Pseudomonas arvilla. The molecular weight of the enzyme was determined to be 38,300 by sedimentation equilibrium analysis, 37,000 by Sephadex G-100 gel filtration, and 37,500 by sodium dodecyl sulfate disc gel electrophoresis, respectively, indicating that the enzyme consisted of a single polypeptide chain. The sedimentation coefficient was calculated to be 3.3 S. The Stokes radius for the enzyme was calculated to be 27 A. The isoelectric point of the enzyme was estimated to be pH 4.2. The enzyme contained 1 mol of FAD, 2 mol of iron, and 2 mol of labile sulfide/mol of enzyme. It exhibited absorption spectrum with maxima at 273, 340, 402, and 467 nm. Amino acid analysis of the enzyme revealed that it was devoid of tryptophan. The enzyme contained 9 mol of cysteine/mol of enzyme but no disulfide linkage. The turnover number of the enzyme for the NADH-dependent reduction of cytochrome c was 17,100 at 24 degrees C. Although NADPH also acted as an electron donor, NADH was highly superior to NADPH. Ferricyanide and 2,6-dichlorophenolindophenol served as electron acceptors. Certain other properties of the enzyme are also presented.