title: Chlamydomonas reinhardtii chloroplasts contain a homodimeric pyruvate:ferredoxin oxidoreductase that functions with FDX1.
abstract: Eukaryotic algae have long been known to live in anoxic environments, but interest in their anaerobic energy metabolism has only recently gained momentum, largely due to their utility in biofuel production. Chlamydomonas reinhardtii figures remarkably in this respect, because it efficiently produces hydrogen and its genome harbors many genes for anaerobic metabolic routes. Central to anaerobic energy metabolism in many unicellular eukaryotes (protists) is pyruvate:ferredoxin oxidoreductase (PFO), which decarboxylates pyruvate and forms acetyl-coenzyme A with concomitant reduction of low-potential ferredoxins or flavodoxins. Here, we report the biochemical properties of the homodimeric PFO of C. reinhardtii expressed in Escherichia coli. Electron paramagnetic resonance spectroscopy of the recombinant enzyme (Cr-rPFO) showed three distinct [4Fe-4S] iron-sulfur clusters and a thiamine pyrophosphate radical upon reduction by pyruvate. Purified Cr-rPFO exhibits a specific decarboxylase activity of 12 µmol pyruvate min⁻¹ mg⁻¹ protein using benzyl viologen as electron acceptor. Despite the fact that the enzyme is very oxygen sensitive, it localizes to the chloroplast. Among the six known chloroplast ferredoxins (FDX1-FDX6) in C. reinhardtii, FDX1 and FDX2 were the most efficient electron acceptors from Cr-rPFO, with comparable apparent K(m) values of approximately 4 µm. As revealed by immunoblotting, anaerobic conditions that lead to the induction of CrPFO did not increase levels of either FDX1 or FDX2. FDX1, being by far the most abundant ferredoxin, is thus likely the partner of PFO in C. reinhardtii. This finding postulates a direct link between CrPFO and hydrogenase and provides new opportunities to better study and engineer hydrogen production in this protist.
authors.affiliation: Unité de Bioénergétique et Ingénierie des Protéines-Unité Mixte de Recherche 7281, Centre National de la Recherche Scientifique-Aix-Marseille Université, 13402 Marseille, France.
authors.forename: Robert
authors.initials: R
authors.lastname: van Lis
authors.forename: Carole
authors.initials: C
authors.lastname: Baffert
authors.forename: Yohann
authors.initials: Y
authors.lastname: Couté
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Nitschke
authors.forename: Ariane
authors.initials: A
authors.lastname: Atteia
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D001594:Benzyl Viologen / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology* / Q000235:genetics; D060365:Chloroplast Proteins / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D004734:Energy Metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005288:Ferredoxins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011767:Pyruvate Decarboxylase / Q000378:metabolism; D050743:Pyruvate Synthase / Q000235:genetics / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012995:Solubility; D013835:Thiamine Pyrophosphate / Q000235:genetics / Q000378:metabolism
chemical_list: D060365:Chloroplast Proteins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D001594:Benzyl Viologen; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D050743:Pyruvate Synthase; D011767:Pyruvate Decarboxylase; D013835:Thiamine Pyrophosphate