The pathway of electron transfer within the nitrogenase complex

Abstract

Site-directed mutagenesis and gene replacement were used to probe the pathway of electron transfer in nitrogenase by substituting single or groups of amino acid residues that, within the current view of component protein docking and nitrogenase catalysis, are likely to be involved in inter- or intra-molecular electron transfer. Intermolecular electron transfer was probed by substituting charged residues that, within the model for component protein docking proposed by Rees and Howard (Kim and Rees. 1992. Nature 360:553-60; Howard. 1993. In Molybdenum Enzymes, Cofactors and Model Systems, eds. EI Stiefel, D Coucouvanis, and WE Newton, pp.271-89. Washington DC: Am. Chem. Soc. 387 pp), are likely to be involved in electrostatic interactions that facilitate component protein association or dissociation. Intramolecular electron transfer was probed by substituting residues which are located in the polypeptide matrix that separates the P cluster and the iron-molybdenum cofactor based on the generally accepted view that the P cluster is an intermediate in electron transfer from the Fe protein to the iron-molybdenum cofactor at the substrate reduction site.

The results of the biochemical characterization of a hybrid Azotobacter vinelandii-Clostridium pasteurianum Fe protein indicate that the region of the A. vinelandii Fe protein defined by residues 59 through 67 is involved in Fe protein-MoFe protein interaction. The rationale for construction of this hybrid Fe protein was based partially on the observation that the Fe protein from C. pasteurianum forms a tight-binding inactive complex with the MoFe protein from A. vinelandii. Detailed studies involving NaCl sensitivity and component protein ratio titrations suggest that this region may have a specific role in component protein dissociation. Further studies involving substitution of individual residues of the MoFe protein indicate that α-Asp¹⁶¹ is involved in component protein interaction.

MoFe protein intramolecular electron transfer was probed by placing amino acid substitutions at β-Tyr⁹⁸, which is located directly between the P cluster and the iron-molybdenum cofactor. The results of the biochemical characterization of an altered MoFe with β-Tyr⁹⁸ substituted by His, support the generally accepted view that electron transfer from the Fe protein to the substrate reduction site involves the P cluster as an intermediate electron acceptor. It was also indicated that the P cluster may be able to accept more than one electron, which is consistent with the mechanism of P cluster reduction suggested by Rees (Rees DC, Chan MK, Kim J. 1993. Adv. Inorg. Chem. 40:89-119).