| Popis: |
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 ChlL2 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(ChlL2)2 complex after incubation with the nonhydrolyzable ATP analogs adenosine 5′-(γ-thio)triphosphate, adenosine 5′-(β,γ-imido)triphosphate, or MgADP in combination with AlF4−. Additionally, a mutant ChlL2 protein, with a deleted Leu153 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 ChlL2, indicating that complete ATP hydrolysis is a prerequisite for intersubunit electron transfer. Circular dichroism spectroscopic experiments indicated nucleotide-dependent conformational changes for ChlL2 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. |
