title: Crystal structure and biochemical characterization of Chlamydomonas FDX2 reveal two residues that, when mutated, partially confer FDX2 the redox potential and catalytic properties of FDX1.
abstract: The green alga Chlamydomonas reinhardtii contains six plastidic [2Fe2S]-cluster ferredoxins (FDXs), with FDX1 as the predominant isoform under photoautotrophic growth. FDX2 is highly similar to FDX1 and has been shown to interact with specific enzymes (such as nitrite reductase), as well as to share interactors with FDX1, such as the hydrogenases (HYDA), ferredoxin:NAD(P) reductase I (FNR1), and pyruvate:ferredoxin oxidoreductase (PFR1), albeit performing at low catalytic rates. Here we report the FDX2 crystal structure solved at 1.18 Å resolution. Based on differences between the Chlorella fusca FDX1 and C. reinhardtii FDX2 structures, we generated and purified point-mutated versions of the FDX2 protein and assayed them in vitro for their ability to catalyze hydrogen and NADPH photo-production. The data show that structural differences at two amino acid positions contribute to functional differences between FDX1 and FDX2, suggesting that FDX2 might have evolved from FDX1 toward a different physiological role in the cell. Moreover, we demonstrate that the mutations affect both the midpoint potentials of the FDX and kinetics of the FNR reaction, possibly due to altered binding between FDX and FNR. An effect on H2 photo-production rates was also observed, although the kinetics of the reaction were not further characterized.
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Marko
authors.initials: M
authors.lastname: Boehm
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Markus
authors.initials: M
authors.lastname: Alahuhta
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Mulder
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Erin A
authors.initials: EA
authors.lastname: Peden
authors.affiliation: Computational Science Center, National Renewable Energy Laboratory, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Hai
authors.initials: H
authors.lastname: Long
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Roman
authors.initials: R
authors.lastname: Brunecky
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Lunin
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Paul W
authors.initials: PW
authors.lastname: King
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Ghirardi
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, Mail Stop: 3313, 15013 Denver West Parkway, Golden, CO, 80401, USA. alexdubini@yahoo.com.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Dubini
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D016825:Chlamydomonas reinhardtii / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein
chemical_list: D005288:Ferredoxins; D010940:Plant Proteins; D009249:NADP; D006859:Hydrogen; D005287:Ferredoxin-NADP Reductase
keywords: Chlamydomonas; Ferredoxin; Hydrogen photo-production; Interaction; NADPH; Structure