Substrate recognition of nitrogenase-like dark operative protochlorophyllide oxidoreductase from Prochlorococcus marinus.
Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL(2) dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB)(2) complex exhibited EPR signals for two [4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB)(2). For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.
Bröcker
Markus J
MJ
Institut für Mikrobiologie, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany.
Wätzlich
Denise
D
Uliczka
Frank
F
Virus
Simone
S
Saggu
Miguel
M
Lendzian
Friedhelm
F
Scheer
Hugo
H
Rüdiger
Wolfhart
W
Moser
Jürgen
J
Jahn
Dieter
D
D002384:Catalysis; D016000:Cluster Analysis; D000458:Cyanobacteria / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D009591:Nitrogenase / Q000235:genetics* / Q000502:physiology*; D010088:Oxidoreductases / Q000378:metabolism / Q000502:physiology*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000378:metabolism / Q000502:physiology*; D048929:Prochlorococcus / Q000201:enzymology*; D011521:Protochlorophyllide / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013379:Substrate Specificity; D013696:Temperature
D011758:Pyrroles; D011521:Protochlorophyllide; D010088:Oxidoreductases; D009591:Nitrogenase; D044925:Oxidoreductases Acting on CH-CH Group Donors; C533890:dark operative protochlorophyllide oxidoreductase, Prochlorococcus marinus