Biochemical characterization of a singular mutant of nitrate reductase from Chlamydomonas reinhardii. New evidence for a heteropolymeric enzyme structure

Abstract

A singular mutant strain from Chlamydomohas reinhardii defective in nitrate reductase has been characterized. Mutant 301 possesses an ammonia-repressible NAD(P)H-cytochrome c reductase with the same charge and size properties as the low molecular weight ammonia-repressible diaphorase present in the wild-type strain 6145 c and is also able to reconstitute NAD(P)H-nitrate reductase activity by in vitro complementation with reduced benzyl viologen-nitrate reductase from mutant 305. Furthermore, a heat-labile costitutive molybdenum cofactor which is fuctionally active is also present in mutant 301. Mutant 301 has the two requirements exhibited by the active nitrate reductase complex from fungi, namely, NAD(P)H-cytochrome c reductase activity and molybdenum cofactor, but lacks NAD(P)H-nitrate reductase activity. This fact together with biochemical data presented from other C. reinhardii mutants strongly suggest a heteropolymeric model for the nitrate reductase complex of the alga.