Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex
Proceedings of the National Academy of Sciences of the United States of America
Photosynthesis uses chlorophylls for the conversion of light into chemical energy, the driving force of life on Earth. During chlorophyll biosynthesis in photosynthetic bacteria, cyanobacteria, green algae and gymnosperms, dark-operative protochlorophyllide oxidoreductase (DPOR), a nitrogenase-like metalloenzyme, catalyzes the chemically challenging two-electron reduction of the fully conjugated ring system of protochlorophyllide a. The reduction of the C-17=C-18 double bond results in the
... cteristic ring architecture of all chlorophylls, thereby altering the absorption properties of the molecule and providing the basis for light-capturing and energytransduction processes of photosynthesis. We report the X-ray crystallographic structure of the substrate-bound, ADP-aluminium fluoride-stabilized (ADP·AlF 3 -stabilized) transition state complex between the DPOR components L 2 and (NB) 2 from the marine cyanobacterium Prochlorococcus marinus. Our analysis permits a thorough investigation of the dynamic interplay between L 2 and (NB) 2 . Upon complex formation, substantial ATP-dependent conformational rearrangements of L 2 trigger the protein-protein interactions with (NB) 2 as well as the electron transduction via redox-active [4Fe-4S] clusters. We also present the identification of artificial "small-molecule substrates" of DPOR in correlation with those of nitrogenase. The catalytic differences and similarities between DPOR and nitrogenase have broad implications for the energy transduction mechanism of related multiprotein complexes that are involved in the reduction of chemically stable double and/or triple bonds. dynamic switch protein | electron transfer T he biosynthesis of chlorophylls is essential for the capture of global energy. This complex, multienzymatic process generates chlorophyllide a (Chlide) through the stereospecific reduction of the C-17=C-18 double bond of ring D in protochlorophyllide a (Pchlide) (Fig. 1A) . Two completely unrelated enzymes have evolved for Pchlide reduction: a monomeric, lightdependent system (1), found in angiosperms and cyanobacteria, and the dark-operative protochlorophyllide oxidoreductase (DPOR), found in anoxygenic photosynthetic bacteria, cyanobacteria, algae, and gymnosperms (2). DPOR is a two-component metalloprotein comprising an ATP-dependent reductase (L 2 ) and a catalytic unit [(NB) 2 ], both sharing a substantial degree of structural and sequence identity with nitrogenase ( Fig. 1B) (3, 4) . As in nitrogenase, both components of DPOR carry redox active metallocenters (5-8), which mediate the ATPdriven electron transfer from L 2 to the site of substrate reduction in (NB) 2 . L 2 and (NB) 2 are only transiently associated with each other during catalysis (9), and ATP hydrolysis triggers their association and dissociation, permitting control of the timing of the accompanying electron transfer process between the two proteins. Previously determined structures of L 2 and (NB) 2 (5-7) provided a static picture of DPOR catalysis. However, only the structural investigation of the "trapped" ternary transition state complex allows for the molecular understanding of DPOR protein dynamics and subcomplex interaction. Results and Discussion The crystal structure determination of the 360 kDa DPOR complex from the marine cyanobacterium Prochlorococcus marinus (10) is summarized in Table S1 , and the heterooctameric complex is depicted in Fig. 1 . Subunits N and B are structurally homologous, generating a pseudo-twofold symmetry axis that is colinear with the molecular twofold axis of L 2 (Fig. 1C) . Both [4Fe-4S] clusters are centered around this extended axis: the L 2 cluster is symmetrically ligated by four cysteinyl ligands between the two subunits, whereas the NB cluster is asymmetrically ligated by three cysteine residues from N and one aspartate residue from B. Apparently, the docking of L 2 on NB induces a linear arrangement of the two redox-active clusters with the substrate, Pchlide (Fig. 1D) . Moreover, upon complex formation, the two [4Fe-4S] clusters are brought to a distance of 14.1 Å, which is substantially shorter than the distance calculated on the basis of the noncomplexed structure of (NB) 2 upon theoretical rigid-body docking of L 2 (5). The distance between the NB cluster and the conjugated Pchlide ring system, by contrast, remains constant at ∼11 Å both in the absence and presence of L 2 . Clearly, electron transfer in DPOR is mediated through the spatial organization of the redox-active [4Fe-4S] centers and the substrate. Biochemical studies identified L 2 as a dynamic switch protein (9) that links ATP hydrolysis to conformational changes in the protein. Two different states of the L 2 protein were characterized biochemically: the "on state" is induced by ATP analogs and has a high affinity for (NB) 2 ; the "off state" is generated in the presence of ADP and does not form a complex with (NB) 2 . The existence of the two states of L 2 is further supported by structural comparison of L 2 within the octameric complex with the free form of a related L 2 protein and the free or complexed form of NifH 2 of nitrogenase (Table S2) , which strongly indicates a parallelism between the switch mechanism of DPOR and nitrogenase. Formation of the DPOR complex leads to a more compact overall structure of L 2 . The underlying structural movements can be described as a rotation of the nucleotide-bound L monomers toward the dimer interface (Fig. 2) , which reveals a striking degree of sequence conservation (Fig. S1 ). The pronounced intersubunit wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The crystallography, atomic coordinates, and structure factors coordinates have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2YNM).