Substrate specificity and evolutionary implications of a NifDK enzyme carrying NifB-co at its active site

Abstract

The in vitro reconstitution of molybdenum nitrogenase was manipulated to generate a chimeric enzyme in which the active site iron–molybdenum cofactor (FeMo-co) is replaced by NifB-co. The NifDK/NifB-co enzyme was unable to reduce N 2 to NH 3, while exhibiting residual C 2H 4 and considerable H 2 production activities. Production of H 2 by NifDK/NifB-co was stimulated by N 2 and was dependent on NifH and ATP hydrolysis. Thus, NifDK/NifB-co is a useful tool to gain insights into the catalytic mechanism of nitrogenase. Furthermore, phylogenetic analysis of D and K homologs indicates that several early emerging lineages, which contain NifB, NifH and NifDK encoding genes but which lack other genes required for processing NifB-co into FeMo-co, might encode an enzyme with similar catalytic properties to NifDK/NifB-co.