Abstract
7-Hydroxymethyl chlorophyll a reductase (HCAR) catalyzes the second half-reaction in chlorophyll b to chlorophyll a conversion. HCAR is required for the degradation of light-harvesting complexes and is necessary for efficient photosynthesis by balancing the chlorophyll a/b ratio. Reduction of the hydroxymethyl group uses redox cofactors [4Fe-4S] cluster and FAD to transfer electrons and is difficult because of the strong carbon-oxygen bond. Here, we report the crystal structure of Arabidopsis HCAR at 2.7-Å resolution and reveal that two [4Fe-4S]clusters and one FAD within a very short distance form a consecutive electron pathway to the substrate pocket. In vitro kinetic analysis confirms the ferredoxin-dependent electron transport chain, thus supporting a proton-activated electron transfer mechanism. HCAR resembles a partial reconstruction of an archaeal F420-reducing [NiFe] hydrogenase, which suggests a common mode of efficient proton-coupled electron transfer through conserved cofactor arrangements. Furthermore, the trimeric form of HCAR provides a biological clue of its interaction with light-harvesting complex II.
Highlights
Hydroxymethyl chlorophyll a reductase (HCAR) Is a Trimer—The purified mature Arabidopsis HCAR is of 49 kDa, and the apparent molecular mass according to size exclusion chromatography (SEC) elution profile is 195 Ϯ 20 kDa (Fig. 1, B and C), which suggests a trimeric or tetrameric state
The detailed mechanism of proton-coupled electron transfer (PCET) is currently under investigation, its fundamental principle is the coupling of electron and proton transfer to cross the intermediate energy barrier [49]
The HCAR-catalyzed hydroxymethyl reduction, in which a resonance-stabilized carbocation could exist as intermediate [10, 50], employs proton-activated electron transfer, a type of PCET occurring in the Q-cycle of the cytochrome bc1 complex [51]
Summary
Protein Expression and Purification—The gene of Arabidopsis HCAR (At1g04620) lacking sequence of the N-terminal 26 amino acid peptide was amplified by PCR. The product was inserted into pETMALc-H [23] with an introduced tobacco etch virus (TEV) cleavage sequence following the MBP-His tag JUNE 17, 2016 VOLUME 291 NUMBER 25
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