Abstract
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'-(gamma-thio)triphosphate, adenosine 5'-(beta,gamma-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.
Highlights
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
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(ChlL2)2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5-(␥-thio)triphosphate, adenosine 5-(,␥-imido)triphosphate, or MgADP in combination with AlF4؊
Electron paramagnetic resonance spectroscopy of ternary DPOR complexes revealed a reduced [4Fe-4S] cluster located on ChlL2, indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer
Summary
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. DPOR Enzyme Assay—The standard DPOR activity assay containing 13 M Pchlide, 2 mM dithionite, as an artificial electron donor, 2 mM ATP, and an ATP regenerating system was performed and analyzed as described earlier in the presence of 100 pmol of purified (ChlN/ChlB)2 and 200 pmol of ChlL2 (125 l final volume) [15, 18]. Control assays lacking subcomplexes of ChlL2 or (ChlN/ChlB)2, control reactions for which the DPOR subunits had been previously inactivated by oxygen exposure or controls in the absence of any protein components containing sole lysis buffer, were carried out to determine spontaneous ATP hydrolysis under conditions of the assay.
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