title: Electron transfer in nitrogenase analyzed by Marcus theory: evidence for gating by MgATP.
abstract: Nitrogenase-catalyzed substrate reduction reactions require electron transfer between two component proteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein, in a reaction that is coupled to the hydrolysis of MgATP. In the present work, electron transfer (Marcus) theory has been applied to nitrogenase electron transfer reactions to gain insights into possible roles for MgATP in this reaction. Evidence is presented indicating that an event associated with either MgATP binding or hydrolysis acts to gate electron transfer between the two component proteins. In addition, evidence is presented that the reaction mechanism can be fundamentally changed such that electron transfer becomes rate-limiting by the alteration of a single amino acid within the nitrogenase Fe protein (deletion of Leu 127, L127 Delta). These studies utilized the temperature dependence of intercomponent electron transfer within two different nitrogenase complexes: the wild-type nitrogenase complex that requires MgATP for electron transfer and the L127 Delta Fe protein-MoFe protein complex that does not require MgATP for electron transfer. It was found that the wild-type nitrogenase electron transfer reaction did not conform to Marcus theory, suggesting that an adiabatic event associated with MgATP interaction precedes electron transfer and is rate-limiting. Application of transition state theory provided activation parameters for this rate-limiting step. In contrast, electron transfer from the L127 Delta Fe protein variant to the MoFe protein (which does not require MgATP hydrolysis) was found to be described by Marcus theory, indicating that electron transfer was rate-limiting. Marcus parameters were determined for this reaction with a reorganization energy (lambda) of 2.4 eV, a coupling constant (HAB) of 0.9 cm-1, a free energy change (Delta G' degrees ) of -22.0 kJ/mol, and a donor-acceptor distance (r) of 14 A. These values are consistent with parameters deduced for electron transfer reactions in other protein-protein systems where electron transfer is rate-limiting. Finally, the electron transfer reaction within the L127 Delta Fe protein-MoFe protein complex was found to be reversible. These results are discussed in the context of models for how MgATP interactions might be coupled to electron transfer in nitrogenase.
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
authors.forename: W N
authors.initials: WN
authors.lastname: Lanzilotta
authors.forename: V D
authors.initials: VD
authors.lastname: Parker
authors.forename: L C
authors.initials: LC
authors.lastname: Seefeldt
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000201:enzymology / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004789:Enzyme Activation; D006868:Hydrolysis; D015640:Ion Channel Gating / Q000235:genetics; D007700:Kinetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008956:Models, Chemical; D008983:Molybdoferredoxin / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D008983:Molybdoferredoxin; D000255:Adenosine Triphosphate; D009591:Nitrogenase; D007930:Leucine