Requirement of NifX and Other Nif Proteins for in vitro Biosynthesis of the Iron-Molybdenum Cofactor of Nitrogenase

Abstract

The iron-molybdenum cofactor (FeMo-co) (a unique prosthetic group that contains molybdenum, iron, sulfur and homocitrate in a ratio of 1:7:9:1), is the site for substrate reduction of nitrogenase (Hoover et al, 1989; Kim, Rees, 1992). The products of at least six nitrogen fixation (nif) genes, including nifQ, nifV, nifB, nifH, nifN and nifE, are required for the biosynthesis of FeMo-co. It has been suggested that NifNE protein might serve as a scaffold for FeMo-co biosynthesis. This hypothesis is supported by NifB-co (the Fe-S precursor of FeMo-co) binding to NifNE in a 2:1 ratio (Roll et al, 1995), but intermediates containing heterometal are not observed on NifNE. In vitro FeMo-co synthesis requires an ATP-regenerating system, molybdate, homocitrate, and at least NifB, NifNE, and NifH proteins. Using in vitro FeMo-co synthesis, the product of NifB was purified (Shah et al, 1994) as a detergent-soluble, small molecule termed NifB-cofactor (NifB-co). The requirement for NifB in the in vitro FeMo-co synthesis assay is satisfied by the addition of NifB-co (Shah et al, 1994) which is an iron-sulfur precursor of FeMo-co. Unfortunately all attempts to gain insights into the NifB-co by various spectroscopic techniques (EPR, MCD, Resonance Raman) have failed to reveal any information about this unusual Fe-S cluster (Shah, Johnson unpublished).