NMR Study of the Electron Transfer Complex of Plant Ferredoxin and Sulfite Reductase

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 proteinprotein 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 sitespecific 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 Plant sulfite reductase (SiR) 2 (EC 1.8.7.1) plays an essential role in the reductive assimilation of sulfate by plants, algae, and cyanobacteria by catalyzing the six-electron reduction of sulfite to sulfide (1). The enzyme is a soluble, monomeric protein with a molecular mass of ϳ65 kDa and contains a single siroheme and a single [4Fe-4S] cluster as prosthetic groups (2, 3). Plant-type ferredoxin (Fd) is a small (11 kDa), one-electron carrier protein with a single [2Fe-2S] cluster and serves as the physiological electron donor for SiR (4). The midpoint redox potential (E m ) values for the [4Fe-4S] cluster and siroheme in maize SiR are Ϫ400 and Ϫ285 mV, respectively (5). Therefore, electron flow to the enzyme from the reduced Fd, which has an Em value ϳ Ϫ400 mV, is thermodynamically favorable A transient electron transfer complex is formed between Fd and SiR with 1:1 stoichiometry to facilitate efficient intermolecular electron transfer (6, 7). Other Fd-dependent enzymes of varying molecular size, primary structure, and prosthetic group composition, such as Fd-NADP ϩ reductase (FNR), nitrite reductase, glutamate synthase, andFd-thioredoxin reductase, also form productive electron transfer complexes with Fd. Several lines of evidence obtained from chemical modification experiments, cross-linking experiments, and mutagenesis experiments have indicated that the complexes between Fd and these Fd-dependent enzymes are mainly stabilized by electrostatic forces through the negative charges of Fd and positive charges of each enzyme (8). An example can be seen in the recently determined crystal structure of the complex between maize leaf Fd and FNR (9). The redox centers of Fd and FNR are in close proximity in the complex to allow fast electron transfer between them. The intermolecular contact site near th...

Physicochemical nature of interfaces controlling ferredoxin NADP+ reductase activity through its interprotein interactions with ferredoxin

“…Detailed studies on enzyme activities and electron transfer rates based on the static and dynamic structures determined by X-ray crystallography and solution NMR spectroscopy have increased our understanding of the molecular mechanisms underlying electron flow and enzymatic activities in photosynthesis and respiration [3,[9][10][11][12]. Solution NMR spectroscopy and calorimetry have provided further information on the dynamic structures of proteins and molecular origins of intermolecular interactions for protein functions [3,13].…”

“…Fd is considered to be a good model protein because it transfers an electron obtained from photosystem I to several target enzymes, including ferredoxin-NADP + reductase (FNR), sulfite reductase (SiR), nitrite reductase (NiR), and hydrogenase [17], through the formation of electron transfer protein complexes [9,11,18]. Fd is a small acidic protein (~10.5 kDa) that accommodates the redox center of the [2Fe-2S] cluster [5].…”

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

“…HydA1 Depends on Electrostatics-As already known from interactions with other electron transfer partners (21)(22)(23), PetF tends to form complexes on the basis of electrostatic charge attraction. Protein interactions depending on intermolecular electrostatic contacts are weakened at increasing ionic strength as demonstrated between ferredoxin and ferredoxin-NADPH oxidoreductase (24).…”

“…Interactions between PetF and various redox partners usually involve three polypeptide motifs (28) -Glu 123 in PetF of C. reinhardtii. These three motifs contain highly conserved acidic residues that direct the first stages of complex generation dominated by electrostatic attraction and intermolecular salt bridge formation (22,29,30). Out of all three motifs, eight acidic residues were chosen for site-directed mutagenesis (Fig.…”

Preface: ferredoxin

“…At this time in the Hase laboratory, enzymatic studies were being combined with structural work to unpick the molecular mechanism of electron transfer between ferredoxin and various enzymes, and to identify the basis of ferredoxin specificity (Kurisu et al 2001;Saitoh et al 2006). This special issue contains one such manuscript, with the groups of Yoko Kimata-Ariga and Genji Kurisu collaborating to elucidate the molecular basis for the specific interaction of root type ferredoxin and FNR, which enables efficient electron transfer in the reverse direction to that seen in photosynthesis (Shinohara et al, this issue).…”