Discovery of Evolutionary Divergence of Biological Nitrogen Fixation and Photosynthesis: Fine Tuning of Biogenesis of the NifH and the ChlL by a Peptidyl-Prolyl Cis/Trans Isomerase

Problem statement: Despite the structural and functional similarities between the nitrogenase that performs biological nitrogen fixation reaction and the Dark Protochlorphyllide Oxidoreductase (DPOR) that performs chlorophyll-biosynthesis, attempts to substitute nitrogenase-components with DPOR-components have hitherto failed. This investigation was undertaken to test if Chlamydomonas reinhardtii protochlorophyllide (Pchlide) reductase (ChlL) that shares some structural similarity with Nitrogenase Reductase (NifH) could complement the functions of NifH in biological nitrogen fixation of Azotobacter vinelandii. Approach: Genetic complementation studies were performed to test if the chlL gene and its mutants cloned under transcriptional control of nifH promoter (nifHp) in a broad-host range low copy plasmid pBG1380 could render a Nif⁺ phenotype to NifH-deficient A. vinelandii strains. Results: Expression of ChlL could render Nif⁺ phenotype to NifH-deficient A. vinelandii only in the absence of NifM, a nif-specific PPIase essential for biogenesis of NifH. The ChlL mutants Cys95Thr and Cys129Thr were unable to substitute for NifH. Thus, the conserved cysteine ligands of [4Fe-4S] cluster in ChlL are essential for successful substitution of NifH by ChlL. Since C-termini of NifH and ChlL demonstrated the least similarity and Pro258, a substrate for the PPIase activity of NifM, is located in the C-terminus of NifH, we posited that replacing the C-terminus of NifH with that of ChlL would render NifM-independence to NifH. The NifH-ChlL chimera could support the growth of NifH- and NifM-deficient A. vinelandii in nitrogen limiting conditions implying that it has acquired NifM-independence. Conclusion/Recommendations: Collectively, these observations suggest that NifM, an evolutionarily conserved nif-specific PPIase, could have contributed to the functional divergence of biological nitrogen fixation and photosynthesis during evolution by virtue of its ability to exert opposing effects on structurally similar substrates, ChlL and NifH.