ATP-driven Reduction by Dark-operative Protochlorophyllide Oxidoreductase from Chlorobium tepidum Mechanistically Resembles Nitrogenase Catalysis

Bröcker, Markus J.; Virus, Simone; Ganskow, Stefanie; Heathcote, Peter; Heinz, Dirk W.; Schubert, Wolf‐Dieter; Jahn, Dieter; Moser, Jürgen

doi:10.1074/jbc.m708010200

Cited by 59 publications

(111 citation statements)

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“…Direct treatment of the catalytic (ChlN/ChlB) 2 complex with the reductant dithionite did not result in substrate reduction. However, in the presence of ChlL 2 and ATP DPOR catalysis followed a Michaelis-Menten type kinetic with respect to the co-substrates dithionite and ATP (13). Hence, ChlL 2 containing two ATP binding sites is the functional electron donor leading to a catalytically active (ChlN/ChlB) 2 protein.…”

Section: Resultsmentioning

confidence: 99%

“…The subunit and iron-sulfur cluster composition and substrate recognition of DPOR were recently characterized (13,15,18). However, information regarding ATP-driven subunit interactions and the related electron transfer were still missing.…”

Section: Resultsmentioning

confidence: 99%

“…ATPase Activity of DPOR-The ATP-dependent DPOR system uses a ferredoxin as a natural electron donor (13,30), whereas for in vitro experiments dithionite as an artificial chemical electron donor was used (13). Direct treatment of the catalytic (ChlN/ChlB) 2 complex with the reductant dithionite did not result in substrate reduction.…”

Section: Resultsmentioning

confidence: 99%

“…1B, lane 3). However, all previous attempts to purify the ternary ChlL 2 (ChlN/ChlB) 2 complex failed (13,18). Therefore, a transient interaction responsible for the electron transfer of subunit ChlL 2 to subcomplex (ChlN/ChlB) 2 was proposed (15).…”

Section: Volume 285 • Number 11 • March 12 2010mentioning

confidence: 99%

“…In chlorophyll-synthesizing organisms DPOR is encoded by the chlN, chlB, and chlL genes (10 -12). The corresponding genes for bacteriochlorophyllsynthesizing organisms have been termed bchN, bchB, and bchL (10,13). DPOR subunits ChlN, ChlB, and ChlL share significant amino acid sequence homologies with nitrogenase subunits NifD, NifK, and NifH, respectively (12,14).…”

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Biosynthesis of (Bacterio)chlorophylls

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et al. 2010

Journal of Biological Chemistry

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Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL 2 subunit carrying a [4Fe-4S] cluster transfers electrons to the corresponding heterotetrameric catalytic subunit (ChlN/ChlB) 2 , which also possesses a redox active [4Fe-4S] cluster. To investigate the transient interaction of both subcomplexes and the resulting electron transfer reactions, the ternary DPOR enzyme holocomplex comprising subunits ChlN, ChlB, and ChlL from the cyanobacterium Prochlorococcus marinus was trapped as an octameric (ChlN/ChlB) 2 (ChlL 2 ) 2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5-(␥-thio)triphosphate, adenosine 5-(␤,␥-imido)triphosphate, or MgADP in combination with AlF 4 ؊ . Additionally, a mutant ChlL 2 protein, with a deleted Leu 153 in the switch II region also allowed for the formation of a stable octameric complex. Furthermore, efficient complex formation required the presence of protochlorophyllide. Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL 2 , indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL 2 after ATP binding. A nucleotide-dependent switch mechanism triggering ternary complex formation and electron transfer was concluded. From these results a detailed redox cycle for DPOR catalysis was deduced. Reduction of protochlorophyllide a (Pchlide)2 to chlorophyllide a (Chlide) is a central step in the biosynthesis of chlorophyll and bacteriochlorophyll (1). Two evolutionarily unrelated enzymes are capable of catalyzing the stereospecific two-electron reduction of the C17-C18 double bond of Pchlide (Fig. 1A) (2-4). Monomeric, light-dependent Pchlide oxidoreductase (POR; NADPH Pchlide oxidoreductase, EC 1.3.1.33) drives the NADPH-dependent reduction (5-7) of Pchlide bound in the active site via absorption of light energy. This light dependence of POR catalysis prevents angiosperms from synthesizing chlorophyll in the dark (8, 9). Anoxygenicphotosynthetic bacteria make use of a different ATP-dependent Pchlide reducing system, which is termed the light-independent, dark operative Pchlide oxidoreductase (DPOR), whereas gymnosperms, mosses, ferns, algae, and cyanobacteria utilize both POR and DPOR (3). In chlorophyll-synthesizing organisms DPOR is encoded by the chlN, chlB, and chlL genes (10 -12). The corresponding genes for bacteriochlorophyllsynthesizing organisms have been termed bchN, bchB, and bchL (10, 13). DPOR subunits ChlN, ChlB, and ChlL share significant amino acid sequence homologies with nitrogenase subunits NifD, NifK, and NifH, respectively (12,14).In

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%