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 energy-transduction 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.

Fig. 1.

Catalysis and 3D structure of DPOR complex (L₂)₂(NB)₂. (A) DPOR catalyzes the formation of Chlide through ATP-dependent, stereospecific reduction of the C-17=C-18 double bond of Pchlide ring D. (B) Schematic representation of DPOR (Left) and nitrogenase (Right) complexes. L₂ and NifH₂ (also named Av2) both contain a subunit-bridging [4Fe–4S] cluster, whereas the [4Fe–4S] cluster at the N/B subunit interface of (NB)₂ is located in an analogous position as the [8Fe–7S] P-cluster at the NifD/NifK subunit interface of (NifDK)₂ (Av1). Sequence identities between the subunits are shown in boxes. (C) Structure of the octameric DPOR complex. Subunits N and B (orange and blue, respectively) are responsible for Pchlide binding. Presence of the ATP analog ADP•AlF₃ triggers the binding of two L₂ protein dimers (green). The dyad axis of L₂ is shown as a broken line. The overall (L₂)₂(NB)₂ complex shows perfect symmetry as indicated by the twofold axis (black lenses); subunits of the symmetry-related protomer are marked L′, N′, and B′ and rendered transparent. (D) Cofactors and the substrate of DPOR are highlighted in a transparent surface representation of the octameric DPOR complex. Edge-to-edge distances are indicated.

Fig. 2.

Dynamic switch mechanism of DPOR. (A) Superposition of the “on state” conformation of L₂ in the ADP•AlF₃–stablized complex (green) and the “off state” conformation of L₂ in the ADP-bound state (gray, PDB ID code 3FWY). Nucleotide-dependent conformational rearrangements trigger the affinity of L₂ for the (NB)₂ core (orange and blue). The [4Fe–4S] cluster of L₂ moves ∼3 Å toward the [4Fe–4S] cluster of (NB)₂. Peptide segments undergoing large Cα-rearrangements are marked I (Asp66–Asp70 of switch I), II (Leu154–Cys158 of switch II), and III (loop region Pro118–Gly126). Regions not involved in significant conformational changes are omitted for clarity. The overall movement of each L subunit toward the L₂ dimer interface becomes evident by a 9.4 Å decrease of the distance between the ADP molecules (measured between the N3-atoms of the two adenine bases in the “off state” and the “on state” of L₂). Relevant water molecules are shown as red spheres. (B) Identical superposition after a 45° clockwise rotation. (C) Binding of ADP•AlF₃ to L₂ in the octameric DPOR complex. Residues provided by the second L monomer are indicated by asterisks. The 2F_o–F_c electron density is contoured at 1.5 σ.

Fig. 3.

Protein–protein interaction surfaces for transition state complexes of DPOR and nitrogenase (PDB ID code 1M34). (A) Van der Waals surface of L₂ viewed along the pseudo-twofold rotational symmetry axis from (NB). (B) Van der Waals surface of (NB) viewed along the twofold rotational symmetry axis from L₂. (C) NifH₂ surface (chains E and F) viewed in the same orientation as in A. (D) (NifDK) surface (chains A and B) viewed in the same orientation as in B. Colors are as follows: subunit L chain A (NifH chain F), dark green; subunit L chain B (NifH chain E), light green; subunit N (NifD), orange; subunit B (NifK), blue. Surface areas of residues involved in protein–protein interactions are shown in gray. In all cases, only half of the core tetramer is depicted as one functional unit for clarity. Key secondary structure elements of the docking partner are displayed as cartoon. [4Fe–4S] clusters located on L₂ and NifH₂ are represented by van der Waals spheres. Secondary structural elements exclusive to the docking of DPOR (*1 and *2, both in subunit N) or nitrogenase (#1 in NifD and #2 NifK), as well as the C-terminal extension of DPOR subunit B (‡), are labeled.

Fig. 4.

Stereoview of Pchlide binding in the ADP•AlF₃–stabilized DPOR complex. Three polar amino acid residues are relevant for protonation at C-17 and C-18: residues His394 and Arg48 are located on one B subunit, whereas Asp290′ is provided by the other B subunit. The water molecule in close proximity to C-18 may act as a proton donor, whereas a second water molecule serves as an axial ligand to the central magnesium. The 2F_o–Fc electron density is contoured at 1.0 σ.