Structure of ADP-aluminium fluoride-stabilized protochlorophyllide oxidoreductase complex

Abstract

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 characteristic 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 investi...

Iron-Sulfur Cluster-dependent Catalysis of Chlorophyllide a Oxidoreductase from Roseobacter denitrificans

“…Threedimensional protein structures of DPOR and nitrogenase revealed an oligomeric architecture consisting of heterotetrameric core complexes (N/B) 2 and (NifD/NifK) 2 , respectively, which require the dynamic interplay with the respective homodimeric subcomplexes L 2 and (NifH) 2 (compare Fig. 1) (11,12).…”

“…Accordingly, the final [4Fe-4S] cluster-dependent Pchlide reduction and the specific substrate recognition in the active site of (N/B) 2 of DPOR are unrelated to nitrogenase catalysis. In contrast, identical mechanisms have been elucidated for the initial steps of DPOR and nitrogenase catalysis (2,11).…”

Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a

“…Then the reduction of the peripheral vinyl group at C8 results in the formation of protochlorophyllide a (Pchlide; compound 11; see Subsequently, reduction of the C17/C18 double bond of ring D leads to the formation of the chlorin ring structure of chlorophyllide a (Chlide; compound 12). Two unrelated enzymes have developed for the regio-and stereospecific reduction of the fully conjugated Pchlide ring system: light-dependent protochlorophyllide oxidoreductase performs a light-driven, NADPH-dependent reduction, whereas the ATP-dependent multisubunit enzyme dark-operative protochlorophyllide a oxidoreductase (DPOR) uses nitrogenase-like biochemistry (3)(4)(5)(6).…”

Reduction Processes in Biosynthesis of Chlorophyll Molecules: Chemical Implication of Enzymatically Regio- and Stereoselective Hydrogenations in the Late Stages of Their Biosynthetic Pathway

“…In contrast, chlorophyll-c [a mixture of c 1 (8-ethyl) cyanobacterium was crystallographically revealed with (divinyl)protochlorophyllide-a (2.6 ¡ resolution). 34 In the complex, the COOH of aspartic acid 290 of ChlB and the water molecule hydrogen-bonded with the 17 2 -COOH of the substrate were proposed as the proton donors for the C17 and C18, respectively.…”