Abstract
Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide. Ferredoxin and sulfite reductase form an electrostatically stabilized 1:1 complex for the intermolecular electron transfer. The protein-protein interaction between these proteins from maize leaves was analyzed by nuclear magnetic resonance spectroscopy. Chemical shift perturbation and cross-saturation experiments successfully mapped the location of two major interaction sites of ferredoxin: region 1 including Glu-29, Glu-30, and Asp-34 and region 2 including Glu-92, Glu-93, and Glu-94. The importance of these two acidic patches for interaction with sulfite reductase was confirmed by site-specific mutation of acidic ferredoxin residues in regions 1 and 2, separately and in combination, by which the ability of mutant ferredoxins to transfer electrons and bind to sulfite reductase was additively lowered. Taken together, this study gives a clear illustration of the molecular interaction between ferredoxin and sulfite reductase. We also present data showing that this interaction surface of ferredoxin significantly differs from that when ferredoxin-NADP(+) reductase is the interaction partner.
Highlights
Plant ferredoxin serves as the physiological electron donor for sulfite reductase, which catalyzes the reduction of sulfite to sulfide
Chemical Shift Perturbation Experiment—A series of 1H-15N transverse-relaxation optimized spectroscopy (TROSY) heteronuclear single quantum correlation (HSQC) spectra of 2H, 15N-labeled Fd was measured as a function of the molar ratios of non-labeled sulfite reductase (SiR) to Fd
A general broadening of the resonance signals that should have occurred upon addition of SiR was minimized by the TROSY effect, and concomitant chemical shift changes were well observed only for certain residues
Summary
Preparation—Sample Fd isoprotein from maize leaf (Fd I) [15] was used in this study. Site-specific mutants of Fd, E29Q/E30Q/D34N, E92Q/E93Q, and E29Q/E30Q/D34N/E92Q/E93Q, were prepared with the QuikChange site-directed mutagenesis kit (Stratagene). Wild-type and mutant molecules of maize Fds were expressed in Escherichia coli strain JM109 cells and purified as described previously [16]. 2H-, 15N-labeled Fd was obtained by the same procedure, except that medium was prepared with 99% D2O containing [2H6]-D-glucose and 15NH4Cl. Maize SiR was expressed in E. coli and purified as described previously [3]. Fds were dissolved in a 50-mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl at the final protein concentrations of 91 and 9.1 M for the measurements in the visible and UV regions, respectively. NMR Spectroscopy—For chemical shift perturbation experiments, 2H, 15N-labeled or 15N-labeled Fd proteins were dissolved at a concentration of 0.1– 0.2 mM in a 30-mM potassium phosphate buffer (pH 7.0) containing 25 mM NaCl and 10% D2O. The reaction was initiated by adding NADPH (0.2 mM), and the oxidation of NADPH was monitored by the decrease at A340
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