title: Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure.
abstract: NADH-dependent reduced ferredoxin:NADP oxidoreductase (NfnAB) is found in the cytoplasm of various anaerobic bacteria and archaea. The enzyme reversibly catalyzes the endergonic reduction of ferredoxin with NADPH driven by the exergonic transhydrogenation from NADPH onto NAD(+). Coupling is most probably accomplished via the mechanism of flavin-based electron bifurcation. To understand this process on a structural basis, we heterologously produced the NfnAB complex of Thermotoga maritima in Escherichia coli, provided kinetic evidence for its bifurcating behavior, and determined its x-ray structure in the absence and presence of NADH. The structure of NfnAB reveals an electron transfer route including the FAD (a-FAD), the [2Fe-2S] cluster of NfnA and the FAD (b-FAD), and the two [4Fe-4S] clusters of NfnB. Ferredoxin is presumably docked onto NfnB close to the [4Fe-4S] cluster distal to b-FAD. NAD(H) binds to a-FAD and NADP(H) consequently to b-FAD, which is positioned in the center of the NfnAB complex and the site of electron bifurcation. Arg(187) is hydrogen-bonded to N5 and O4 of the bifurcating b-FAD and might play a key role in adjusting a low redox potential of the FADH(•)/FAD pair required for ferredoxin reduction. A mechanism of FAD-coupled electron bifurcation by NfnAB is proposed.
authors.affiliation: From the Max-Planck-Institut für Biophysik, D-60438 Frankfurt am Main, Germany and the Max-Planck-Institut für Terrestrische Mikrobiologie, D-35043 Marburg, Germany.
authors.forename: Julius K
authors.initials: JK
authors.lastname: Demmer
authors.affiliation: the Max-Planck-Institut für Terrestrische Mikrobiologie, D-35043 Marburg, Germany.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Huang
authors.affiliation: the Max-Planck-Institut für Terrestrische Mikrobiologie, D-35043 Marburg, Germany.
authors.forename: Shuning
authors.initials: S
authors.lastname: Wang
authors.affiliation: From the Max-Planck-Institut für Biophysik, D-60438 Frankfurt am Main, Germany and.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Demmer
authors.affiliation: the Max-Planck-Institut für Terrestrische Mikrobiologie, D-35043 Marburg, Germany thauer@mpi-marburg.mpg.de.
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Thauer
authors.affiliation: From the Max-Planck-Institut für Biophysik, D-60438 Frankfurt am Main, Germany and ulrich.ermler@biophys.mpg.de.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Ermler
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004579:Electron Transport; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism; D005415:Flavins / Q000737:chemistry / Q000378:metabolism*; D006860:Hydrogen Bonding; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009243:NAD / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D005288:Ferredoxins; D005415:Flavins; D007506:Iron-Sulfur Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D009243:NAD; D005287:Ferredoxin-NADP Reductase
keywords: NADH-dependent reduced ferredoxin-NADP oxidoreductase; crystal structure; electron bifurcation; energy metabolism; enzyme catalysis; flavin; iron-sulfur protein