title: Iron-sulfur cluster-dependent catalysis of chlorophyllide a oxidoreductase from Roseobacter denitrificans.
abstract: Bacteriochlorophyll a biosynthesis requires the stereo- and regiospecific two electron reduction of the C7-C8 double bond of chlorophyllide a by the nitrogenase-like multisubunit metalloenzyme, chlorophyllide a oxidoreductase (COR). ATP-dependent COR catalysis requires interaction of the protein subcomplex (BchX)2 with the catalytic (BchY/BchZ)2 protein to facilitate substrate reduction via two redox active iron-sulfur centers. The ternary COR enzyme holocomplex comprising subunits BchX, BchY, and BchZ from the purple bacterium Roseobacter denitrificans was trapped in the presence of the ATP transition state analog ADP·AlF4(-). Electron paramagnetic resonance experiments revealed a [4Fe-4S] cluster of subcomplex (BchX)2. A second [4Fe-4S] cluster was identified on (BchY/BchZ)2. Mutagenesis experiments indicated that the latter is ligated by four cysteines, which is in contrast to the three cysteine/one aspartate ligation pattern of the closely related dark-operative protochlorophyllide a oxidoreductase (DPOR). In subsequent mutagenesis experiments a DPOR-like aspartate ligation pattern was implemented for the catalytic [4Fe-4S] cluster of COR. Artificial cluster formation for this inactive COR variant was demonstrated spectroscopically. A series of chemically modified substrate molecules with altered substituents on the individual pyrrole rings and the isocyclic ring were tested as COR substrates. The COR enzyme was still able to reduce the B ring of substrates carrying modified substituents on ring systems A, C, and E. However, substrates with a modification of the distantly located propionate side chain were not accepted. A tentative substrate binding mode was concluded in analogy to the related DPOR system.
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Svenja
authors.initials: S
authors.lastname: Kiesel
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Denise
authors.initials: D
authors.lastname: Wätzlich
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Christiane
authors.initials: C
authors.lastname: Lange
authors.affiliation: Max-Planck-Institute for Chemical Energy Conversion, D-45470 Mülheim, Germany.
authors.forename: Edward
authors.initials: E
authors.lastname: Reijerse
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University New Haven, Connecticut 06520, and.
authors.forename: Markus J
authors.initials: MJ
authors.lastname: Bröcker
authors.affiliation: Department Biology I, Botany, Ludwig-Maximilians-Universität München, D-80638 München, Germany.
authors.forename: Wolfhart
authors.initials: W
authors.lastname: Rüdiger
authors.affiliation: Max-Planck-Institute for Chemical Energy Conversion, D-45470 Mülheim, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Lubitz
authors.affiliation: Department Biology I, Botany, Ludwig-Maximilians-Universität München, D-80638 München, Germany.
authors.forename: Hugo
authors.initials: H
authors.lastname: Scheer
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany, j.moser@tu-bs.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moser
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
mesh_terms: D002735:Chlorophyllides / Q000737:chemistry / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry; D010788:Photosynthesis / Q000235:genetics*; D041904:Roseobacter / Q000201:enzymology* / Q000235:genetics
chemical_list: D002735:Chlorophyllides; C034388:chlorophyllide a; D010088:Oxidoreductases; D005287:Ferredoxin-NADP Reductase; D009591:Nitrogenase; D003545:Cysteine
keywords: COR; Chlorophyllide Oxidoreductase; DPOR; Dark-operative Protochlorophyllide Oxidoreductase; Electron Paramagnetic Resonance (EPR); Electron Transport; Iron-Sulfur Protein; Nitrogenase; Photosynthetic Pigment