Abstract
In Azotobacter vinelandii, deletion of the fdxA gene, which encodes ferredoxin I (FdI), leads to activation of the expression of the fpr gene, which encodes NADPH-ferredoxin reductase (FPR). In order to investigate the relationship of these two proteins further, the interactions of the two purified proteins have been examined. AvFdI forms a specific 1:1 cross-linked complex with AvFPR through ionic interactions formed between the Lys residues of FPR and Asp/Glu residues of FdI. The Lys in FPR has been identified as Lys258, a residue that forms a salt bridge with one of the phosphate oxygens of FAD in the absence of FdI. UV-Vis and circular dichroism data show that on binding FdI, the spectrum of the FPR flavin is hyperchromatic and red-shifted, confirming the interaction region close to the FAD. Cytochrome c reductase assays and electron paramagnetic resonance data show that electron transfer between the two proteins is pH-dependent and that the [3Fe-4S]+ cluster of FdI is specifically reduced by NADPH via FPR, suggesting that the [3Fe-4S] cluster is near FAD in the complex. To further investigate the FPR:FdI interaction, the electrostatic potentials for each protein were calculated. Strongly negative regions around the [3Fe-4S] cluster of FdI are electrostatically complementary with a strongly positive region overlaying the FAD of FPR, centered on Lys258. These proposed interactions of FdI with FPR are consistent with cross-linking, peptide mapping, spectroscopic, and electron transfer data and strongly support the suggestion that the two proteins are physiological redox partners.
Highlights
Azotobacter vinelandii ferredoxin I (AvFdI)1 is a small ironsulfur ([Fe-S]) protein that has been extensively characterized by x-ray crystallography and direct electrochemical and spectroscopic methods [1,2,3,4,5,6]
It has been known for some time that FdI has a metabolic function unrelated to nitrogen fixation that is important for cell growth [14], the specific cellular function of FdI and related seven-iron ferredoxins in other organisms has yet to be determined
In order to investigate the mechanism of regulation by FdI, the small acidic protein that is overexpressed in response to fdxA deletion was purified and characterized [15], and the gene encoding the protein was cloned and sequenced [16]
Summary
Azotobacter vinelandii ferredoxin I (AvFdI)1 is a small ironsulfur ([Fe-S]) protein that has been extensively characterized by x-ray crystallography and direct electrochemical and spectroscopic methods [1,2,3,4,5,6]. We did a series of experiments in which we chemically modified either the lysine or the glutamate/aspartate residues in FdI and/or FPR prior to attempting to form the cross-linked complex with EDC.
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