Identification of residues of Rhodobacter capsulatus ferredoxin I important for its interaction with nitrogenase.
In Rhodobacter capsulatus, ferredoxin I (FdI) serves as natural electron donor to nitrogenase. In order to probe amino acid residues possibly involved in the interaction with dinitrogenase reductase, FdI was subjected to site-specific mutagenesis. A three-dimensional structure of FdI was designed by computer modelling and used for selecting target residues. Mutant ferredoxins bearing substitutions of surface residues, as well as a variant having a Met2 --> Tyr replacement in the vicinity of one cluster, have been constructed. All FdI variants were expressed to similar levels both in Escherichia coli and in a FdI-deleted mutant of the natural host. Once purified, the mutant ferredoxins exhibited molecular and spectroscopic properties almost identical to wild-type FdI. Determination of the reduction potential of FdI by cyclic voltammetry gave an E'o of -510 mV (pH 7.6) for both clusters, which is one of the lowest values reported for a 2[4Fe-4S] ferredoxin. Only the [Tyr2]FdI variant showed a significant difference in redox potential (delta E'o = -15 mV). Based on in vitro assays, a [Glu27, Glu28]FdI double mutant exhibited a twofold decrease in the electron transfer rate to dinitrogenase reductase while the affinity of this mutant for the enzyme was barely affected. On the other hand, an Asp36 --> His substitution resulted in a sevenfold increase of the apparent Km for dinitrogenase reductase. Unlike FdI and the other mutant ferredoxins, the [His36]FdI variant also failed to form a cross-linked complex with dinitrogenase reductase upon incubation with a carbodiimide. It is concluded that Asp36 in FdI probably participates in the interaction between the two protein partners. Nevertheless, all the FdI mutants proved competent in restoring a wild-type phenotype when expressed in a FdI-deleted mutant background, indicating that none of the studied residues was absolutely critical for electron transfer to nitrogenase.
Naud
I
I
CNRS URA 1130, Département de Biologie Moléculaire et Structurale, Grenoble, France.
Meyer
C
C
David
L
L
Breton
J
J
Gaillard
J
J
Jouanneau
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003198:Computer Simulation; D003432:Cross-Linking Reagents; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009591:Nitrogenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D010641:Phenotype; D017354:Point Mutation; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D003432:Cross-Linking Reagents; D005288:Ferredoxins; D009591:Nitrogenase