In vitro biosynthesis of iron molybdenum cofactor and maturation of the nif-encoded apodinitrogenase - Effect of substitution for NifH with site-specifically altered forms of NifH

has three different roles in the nitrogenase enzyme system. Apart from serv ing as the physiological electron donor to dinitrogenase, NifH is involved in iron-molybdenum cofactor (FeMo-co) biosynthesis and in maturation of the FeMo-co-deficient form of apodinitrogenase to a FeMo-co-activable form (ap odinitrogenase maturation). The exact roles of NifH in these processes are not well understood. In the present study, the features of NifH required fo r the aforementioned processes have been investigated by the use of site-sp ecifically altered forms of the enzyme, The ability of six altered forms of NifH inactive in substrate reduction (K15R, D39N, D43N, L127 Delta, D129E, and F135Y) to function in in vitro FeMo-co synthesis and apodinitrogenase maturation reactions was investigated. We report that the ability of NifH t o bind and not hydrolyze MgATP is required for it to function in these proc esses. We also present evidence that the ability of NifH to function in the se processes is not dictated by the properties known to be required for its function in electron transfer to dinitrogenase, Evidence toward the existe nce of separate, overlapping sites on NifH for each of its functions (subst rate reduction, FeMo-co biosynthesis, and apodinitrogenase maturation) is p resented.