Catalytic activities of NifEN: implications for nitrogenase evolution and mechanism.
NifEN is a key player in the biosynthesis of nitrogenase MoFe protein. It not only shares a considerable degree of sequence homology with the MoFe protein, but also contains clusters that are homologous to those found in the MoFe protein. Here we present an investigation of the catalytic activities of NifEN. Our data show that NifEN is catalytically competent in acetylene (C(2)H(2)) and azide (N(3)(-)) reduction, yet unable to reduce dinitrogen (N(2)) or evolve hydrogen (H(2)). Upon turnover, C(2)H(2) gives rise to an additional S = 1/2 signal, whereas N(3)(-) perturbs the signal originating from the NifEN-associated FeMoco homolog. Combined biochemical and spectroscopic studies reveal that N(3)(-) can act as either an inhibitor or an activator for the binding and/or reduction of C(2)H(2), while carbon monoxide (CO) is a potent inhibitor for the binding and/or reduction of both N(3)(-) and C(2)H(2). Taken together, our results suggest that NifEN is a catalytic homolog of MoFe protein; however, it is only a "skeleton" version of the MoFe protein, as its associated clusters are simpler in structure and less versatile in function, which, in turn, may account for its narrower range of substrates and lower activities of substrate reduction. The resemblance of NifEN to MoFe protein in catalysis points to a plausible, sequential appearance of the two proteins in nitrogenase evolution. More importantly, the discrepancy between the two systems may provide useful insights into nitrogenase mechanism and allow reconstruction of a fully functional nitrogenase from the "skeleton" enzyme, NifEN.
Hu
Yilin
Y
Department of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900, USA. yilinh@uci.edu
Yoshizawa
Janice M
JM
Fay
Aaron W
AW
Lee
Chi Chung
CC
Wiig
Jared A
JA
Ribbe
Markus W
MW
D000114:Acetylene / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D020134:Catalytic Domain; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D019143:Evolution, Molecular; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008983:Molybdoferredoxin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009591:Nitrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D001386:Azides; D001426:Bacterial Proteins; D008983:Molybdoferredoxin; D002248:Carbon Monoxide; D009591:Nitrogenase; D000114:Acetylene