Genetic analysis of the NifM dependence of the nitrogenase iron proteins

NifH maturation in certain diazotrophic bacteria depends on NifM, a protein with a PpiC-isomerase domain. Intriguingly, the putative proline substrate for NifM is present in NifH proteins from strains lacking nifM, raising questions regarding the actual role of NifM and the necessity of isomerases for the maturation of NifH proteins in organisms devoid of nifM. Here, we established that the C-terminal PpiC-isomerase domain of NifM is predominant in NifH maturation, whereas the N-terminal region may facilitate this process by binding to NifH. Proline scanning indicated that Pro²⁵⁶ of NifH is more likely involved in NifM-directed maturation than the previously proposed Pro²⁵⁸, as NifH variants from Klebsiella oxytoca with P258I, T, or V substitutions retained full activity while still requiring NifM for function. Conversely, alterations of Pro²⁵⁶ to any amino acid resulted in loss of function and insolubility. We subsequently assessed the NifM dependence of 15 NifHs from diverse bacteria based on the reconstituted nitrogenase system in Escherichia coli. Additionally, we validated the NifM independence of a selected NifH from Bacillus in E. coli strains deficient in ppiC, ppiD, and surA genes, both individually and in combination, as these gene products also contain a PpiC-isomerase domain. In conclusion, our findings rectified earlier misconceptions about the catalytic site in NifH for NifM and demonstrated that the PpiC isomerase domain is not required for the maturation of the NifH protein in bacteria without nifM. Moreover, the NifM-independent NifH, identified in this study, has the potential to engineer diazotrophs in heterogeneous hosts.IMPORTANCEThe iron protein NifH is one of the most important components of the nitrogenase system, contributing to cofactor biosynthesis and acting as an obligate electron donor for nitrogenase under in vivo conditions. Unraveling the maturation mechanism of NifH holds substantial significance for both theoretical and practical research. Our investigation into the dependence of NifM on NifH maturation has yielded novel insights into the function of NifM and has corrected previous misconceptions regarding the catalytic site in NifH for NifM. The prospect of engineering diazotrophs in heterologous hosts, particularly crops, through the direct transfer of the nitrogenase system represents an area of considerable scientific interest. Notably, the isomerase-independent NifH identified in this study may offer advantages in heterologous expression scenarios as it requires fewer accessory proteins for functionality.