Role of a Cluster of Hydrophobic Residues Near the FAD Cofactor in Anabaena PCC 7119 Ferredoxin-NADP+Reductase for Optimal Complex Formation and Electron Transfer to Ferredoxin

Marta Martı́Nez-Júlvez,Isabel Nogués,Merche Faro,John K Hurley,Tammy B Brodie,Tomás Mayoral,Julia Sanz-Aparicio,Juan A Hermoso,Marian T Stankovich,Milagros Medina,Gordon Tollin,Carlos Gómez-Moreno

doi:10.1074/jbc.m102112200

Abstract

In the ferredoxin-NADP(+) reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction. We have investigated further the role of hydrophobic interactions in complex stabilization and ET between these proteins by replacing three hydrophobic residues, Leu-76, Leu-78, and Val-136, situated on the FNR surface in the vicinity of its FAD cofactor. Whereas neither the ability of FNR to accept electrons from NADPH nor its structure appears to be affected by the introduced mutations, different behaviors with Fd are observed. Thus, the ET interaction with Fd is almost completely lost upon introduction of negatively charged side chains. In contrast, only subtle changes are observed upon conservative replacement. Introduction of Ser residues produces relatively sizable alterations of the FAD redox potential, which can explain the modified behavior of these mutants. The introduction of bulky aromatic side chains appears to produce rearrangements of the side chains at the FNR/Fd interaction surface. Thus, subtle changes in the hydrophobic patch influence the rates of ET to and from Fd by altering the binding constants and the FAD redox potentials, indicating that these residues are especially important in the binding and orientation of Fd for efficient ET. These results are consistent with the structure reported for the Anabaena FNR.Fd complex.

Highlights

In the ferredoxin-NADP؉ reductase (FNR)/ferredoxin (Fd) system, an aromatic amino acid residue on the surface of Anabaena Fd, Phe-65, has been shown to be essential for the electron transfer (ET) reaction
In the present study we further investigate the role of hydrophobic interactions in complex stabilization and ET between Fd and FNR, by analyzing the function of several hydrophobic residues that the three-dimensional structure of the protein shows are situated on the protein surface in the vicinity of the FAD isoalloxazine ring (Fig. 1A)
Our results clearly indicate that these residues are important for efficient ET and provide additional evidence that the crystallographic structure reported for the Anabaena FNR1⁄7Fd complex [26] represents a viable model for a productive ET complex

Summary

EXPERIMENTAL PROCEDURES

Oligonucleotide-directed Mutagenesis—The FNR mutants were produced using a construct of the petH gene, which had been previously cloned into the expression vector pTrc99a, as a template [20]. FNR species and oxidized ferredoxin were measured by differential absorption spectroscopy as described previously [14]. These experiments were performed on solutions containing ϳ20 ␮M FNR in 50 mM. Samples of reduced Fd and FNR were prepared by photoreduction of the proteinbound redox center in 50 mM Tris/HCl, pH 8.0, containing 20 ␮M. The dRf and EDTA were present to initiate photoreduction of proteins via the highly reductive deazaflavin radical [39]. Alternative reduction methods such as electrochemical mediation by methyl viologen yielded similar results. The atomic coordinates for all mutants have been deposited in the Protein Data Bank with accession codes 1qgz for the L78D model, 1qh0 for the L76D/L78D, and 1h85 for the V136L

RESULTS

88 Ϯ 3 72 Ϯ 2

46 Ϯ 6 42 Ϯ 4

DISCUSSION