Changes in the midpoint potentials of the nitrogenase metal centers as a result of iron protein-molybdenum-iron protein complex formation.
All nitrogenase-catalyzed substrate reduction reactions require the transient association between the iron (Fe) protein component and the molybdenum-iron (MoFe) protein component with concomitant intercomponent electron transfer and MgATP hydrolysis. Understanding the effects of Fe protein-MoFe protein complex formation on the properties of the nitrogenase metal centers is thus essential to understanding the electron transfer reactions. This work presents evidence for significant shifts in midpoint potentials for two of the three nitrogenase metal centers as a result of Fe protein binding to the MoFe protein. The midpoint potentials for the three nitrogenase metal centers, namely the [4Fe-4S] cluster of the Fe protein, and the [8Fe-7S] (or P-) clusters and FeMo cofactors (or M-centers) of the MoFe protein, were determined within a nondissociating nitrogenase complex prepared with a site-specifically altered Fe protein (Leu at position 127 deleted, L127Delta). The midpoint potential for each metal center was determined by mediated redox titrations, with the redox state of each center being monitored by parallel and perpendicular mode EPR spectroscopy. The midpoint potential of the Fe protein [4Fe-4S]2+/1+ cluster couple was observed to change by -200 mV from -420 mV in the uncomplexed L127Delta Fe protein to -620 mV in the L127Delta Fe protein-MoFe protein complex. The midpoint potential of the two electron oxidized couple of the P-clusters (P2+/N) of the MoFe protein was observed to shift by -80 mV upon protein-protein complex formation. No significant change in the midpoint potential of an oxidized state of FeMoco (Mox/N) was observed upon complex formation. These results provide insights into the energetics of intercomponent electron transfer in nitrogenase, suggesting that the energy of protein-protein complex formation is coupled to an increase in the driving force for electron transfer. The results are interpreted in light of the expected changes in the protein environments of the metal centers within the nitrogenase complex.
Lanzilotta
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WN
Department of Chemistry and Biochemistry, Utah State University, Logan, Utah 84322, USA.
Seefeldt
L C
LC
D000255:Adenosine Triphosphate / Q000378:metabolism; D016948:Azotobacter vinelandii / Q000201:enzymology; D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D008961:Models, Structural; D008983:Molybdoferredoxin / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000737:chemistry* / Q000378:metabolism*; D019159:Nonheme Iron Proteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011199:Potentiometry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D008983:Molybdoferredoxin; D019159:Nonheme Iron Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D009591:Nitrogenase