Abstract
Nitrogenase-catalyzed substrate reduction reactions require the association of the iron (Fe) protein and the molybdenum-iron (MoFe) protein, electron transfer from the Fe protein to the MoFe protein coupled to the hydrolysis of MgATP, followed by protein-protein complex dissociation. This work examines the role of MgATP hydrolysis and electron transfer in the dissociation of the Fe protein-MoFe protein complex. Alteration of aspartate 39 to asparagine (D39N) in the nucleotide binding site of Azotobacter vinelandii Fe protein by site-directed mutagenesis resulted in an Fe protein-MoFe protein complex that did not dissociate after electron transfer. While the D39N Fe protein-MoFe protein complex was inactive in all substrate reduction reactions, the complex catalyzed both reductant-dependent and reductant-independent MgATP hydrolysis. Once docked to the MoFe protein, the D39N Fe protein was found to transfer one electron to the MoFe protein requiring MgATP hydrolysis, with an apparent first order rate constant of 0.02 s-1 compared with 140 s-1 for the wild-type Fe protein. Only following electron transfer to the MoFe protein did the D39N Fe protein form a tight complex with the MoFe protein, with no detectable dissociation rate. This was in contrast with the dissociation rate constant of the wild-type Fe protein from the MoFe protein following electron transfer of 5 s-1. Chemically oxidized D39N Fe protein with MgADP-bound did not form a tight complex with the MoFe protein, showing a dissociation rate similar to chemically oxidized wild-type Fe protein (3 s-1 for D39N Fe protein and 6 s-1 for wild-type Fe protein). These results suggest that electron transfer from the Fe protein to the MoFe protein within the protein-protein complex normally induces conformational changes which increase the affinity of the Fe protein for the MoFe protein. A model is presented in which Asp-39 participates in a nucleotide signal transduction pathway involved in component protein-protein dissociation.
Highlights
Nitrogenase-catalyzed substrate reduction reactions require the association of the iron (Fe) protein and the molybdenum-iron (MoFe) protein, electron transfer from the Fe protein to the MoFe protein coupled to the hydrolysis of MgATP, followed by protein-protein complex dissociation
The results from the present study can be discussed in the context of two important questions relating to the signal for dissociation of the nitrogenase proteins. (i) What is the role of MgATP hydrolysis in signaling proteinprotein dissociation, and (ii) what is the role of electron transfer from the Fe protein to the MoFe protein in signaling proteinprotein dissociation?
MgATP binding to the Fe protein clearly induces protein conformational changes that are communicated from the nucleotide binding sites at the center of the protein to the [4Fe-4S] cluster and MoFe protein docking surface approximately 19 Å away [2, 14]
Summary
Iron protein of nitrogenase; MoFe protein, molybdenum-iron protein of nitrogenase; FeMoco, [1Mo7Fe-8S-1homocitrate] cofactor of nitrogenase; P-cluster, [8Fe-(7– 8)S] cluster of nitrogenase; MOPS, 3-(N-morpholino)propanesulfonic acid; IDS; indigo disulfonate. It seemed possible that Asp-39 could be essential for two important functions of the nitrogenase Fe protein, (i) Asp-39 could be a general base, activating a water molecule for nucleophilic attack on the ␥-phosphate of MgATP, and (ii) Asp-39 could function in switch I nucleotide signal transduction to the MoFe protein docking interface. To probe these possible functions of Asp-39, we have changed this residue to the amino acids asparagine (D39N) and glutamic acid (D39E) and characterized the properties of the purified, altered Fe proteins. These results suggest that Asp-39 participates in the signal for component protein dissociation following electron transfer and that electron transfer induces protein conformational changes within the nitrogenase protein-protein complex
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