Isolation and properties of the NAD(P)H-cytochrome c reductase subunit of Chlamydomonas reinhardii NAD(P)H-nitrate reductase

Abstract

Chlamydomonas reinhardii wild strain 6145 c as well as the mutant strain 104, which lacks NAD(P)H-nitrate reductase (EC 1.6.6.2) activity and molybdenum-containing cofactor, exhibit and ammonia-repressible NAD(P)H-cytochrome c reductase activity of identical molecular size, 44 500. This diaphorase activity, which in both strains produces a fast-moving band on electrophoresis, has been separated from other constitutive diphorases unrelated to nitrate reduction by means of Blue-agarose affinity chromatography and characterized as the sole NAD(P)H-diaphorase able to reconstitute the native NAD(P)H-nitrate reductase complex by complementation with extracts of mutant 305, which only has reduced flavin or viologen-nitrate reductase activity. The ammonia-repressible NAD(P)H-cytochrome c reductase possesses FAD and a b-type cytochrome, and its kinetic and enzymatic parameters are similar to those previously reported for the diaphorase activity of the whole complex, which indicates that the NAD(P)H-cytochrome c reductase of 104 mutant is a true subunit of the Chlamydomonas reinhardii NAD(P)H-nitrate reductase native enzyme.