Electrostatic forces involved in orienting Anabaena ferredoxin during binding to Anabaena ferredoxin : NADP(+) reductase: Site-specific mutagenesis, transient kinetic measurements, and electrostatic surface potentials

Transient absorbance measurements following laser flash photolysis have bee n used to measure the rate constants for electron transfer (et) from reduce d Anabaena ferredoxin (Fd) to wild-type and seven site-specific charge-reve rsal mutants of Anabaena ferredoxin:NADP(+) reductase (FNR). These mutation s have been designed to probe the importance of specific positively charged amino acid residues on the surface of the FNR molecule near the exposed ed ge of the FAD cofactor in the protein-protein interaction during et with Fd . The mutant proteins fall into two groups: overall, the K75E, R16E, and K7 2E mutants are most severely impaired in et, and the K138E, R264E, K290E, a nd K294E mutants are impaired to a lesser extent, although the degree of im pairment varies with ionic strength. Binding constants for complex formatio n between the oxidized proteins and for the transient et complexes show tha t the severity of the alterations in et kinetics for the mutants correlate with decreased stabilities of the protein-protein complexes. Those mutated residues, which show the largest effects, are located in a region of the pr otein in which positive charge predominates, and charge reversals have larg e effects on the calculated local surface electrostatic potential. In contr ast, K138, R264, K290, and K294 are located within or close to regions of i ntense negative potential, and therefore die introduction of additional neg ative charges have considerably smaller effects on the calculated surface p otential. We attribute the relative changes in et kinetics and complex bind ing constants for these mutants to these characteristics of the surface cha rge distribution in FNR and conclude that the positively charged region of the FNR surface located in the vicinity of K75, R16, and K72 is especially important in the binding and orientation of Fd during electron transfer.