The cytochrome b 6 f (cytb 6 f) complex plays a central role in oxygenic photosynthesis, linking electron transfer between photosystems I and II and conserving solar energy as a transmembrane proton gradient for ATP synthesis 1-3. Electron transfer within cytb 6 f occurs via the Q-cycle, which catalyses the oxidation of plastoquinol (PQH 2) and the reduction of both plastocyanin (PC) and plastoquinone (PQ) at two separate sites via electron bifurcation 2. In higher-plants cytb 6 f also acts as a redox-sensing hub, pivotal to the regulation of light harvesting a n d c y c l i c e l e c t r o n t r a n s f e r t h at protect against metabolic and environmental stresses 3. Here we present a 3.6 Å resolution c r y o-e l e c t r o n m i c r o s c o p y (c r y o-E M) structure of the dimeric cytb 6 f complex from spinach, which reveals the structural basis for operation of the Q-cycle and its redox sensing function. The complex contains up to three natively bound PQ molecules. The first, PQ1, is bound to one cytb 6 f monomer at the PQ oxidation site (Q p) adjacent to haem b p and chlorophyll a. Two conformations of the chlorophyll a phytyl tail were resolved, one that prevents access to the Q p site and another that permits it, supporting a gating function for the chlorophyll a involved in redox sensing. PQ2 straddles the intermonomer cavity, partially obstructing the PQ reduction site (Q n) on the PQ1 side and committing the electron transfer network to turnover at the occupied Q n site in the neighbouring monomer. A conformational switch involving the haem c n propionate promotes two-electron, two-proton reduction at the Q n site and avoids formation of the reactive intermediate semiquinone. The location of a tentatively assigned third PQ molecule is consistent with a transition between the Q p and Q n sites in opposite monomers during the Q-cycle. The spinach cytb 6 f structure therefore provides new insights into how the complex fulfils its catalytic and regulatory roles in photosynthesis. Photosynthesis sustains life on Earth by converting light into chemical energy in the form of ATP and NADPH, producing oxygen as a by-product. Two light-powered electron transfer reactions at photosystems I and II (PSI and PSII) are linked via the cytb 6 f complex to form the so-called 'Z-scheme' of photosynthetic linear electron transfer (LET) 1. Cytb 6 f catalyses the rate-limiting step in the LET chain, coupling the oxidation of PQH 2 and reduction of PC and PQ to the generation of a transmembrane proton gradient (p), used by ATP synthase to make ATP 2,3. The cytb 6 f complex is analogous to the cytochrome bc 1 (cytbc 1) complex found in mitochondria 4 and anoxygenic photosynthetic bacteria 5 and both operate via the modified Q

The reaction-center light-harvesting complex 1 (RC-LH1) is the core photosynthetic component in purple phototrophic bacteria. We present two cryo–electron microscopy structures of RC-LH1 complexes from Rhodopseudomonas palustris. A 2.65-Å resolution structure of the RC-LH114-W complex consists of an open 14-subunit LH1 ring surrounding the RC interrupted by protein-W, whereas the complex without protein-W at 2.80-Å resolution comprises an RC completely encircled by a closed, 16-subunit LH1 ring. Comparison of these structures provides insights into quinone dynamics within RC-LH1 complexes, including a previously unidentified conformational change upon quinone binding at the RC QB site, and the locations of accessory quinone binding sites that aid their delivery to the RC. The structurally unique protein-W prevents LH1 ring closure, creating a channel for accelerated quinone/quinol exchange.

The X-ray crystal structure of the Rhodopseudomonas (Rps.) palustris reaction center-light harvesting 1 (RC-LH1) core complex revealed the presence of a sixth protein component, variably referred to in the literature as helix W, subunit W or protein W. The position of this protein prevents closure of the LH1 ring, possibly to allow diffusion of ubiquinone/ubiquinol between the RC and the cytochrome bc1 complex in analogous fashion to the well-studied PufX protein from Rhodobacter sphaeroides. The identity and function of helix W have remained unknown for over 13 years; here we use a combination of biochemistry, mass spectrometry, molecular genetics and electron microscopy to identify this protein as RPA4402 in Rps. palustris CGA009. Protein W shares key conserved sequence features with PufX homologs, and although a deletion mutant was able to grow under photosynthetic conditions with no discernible phenotype, we show that a tagged version of protein W pulls down the RC-LH1 complex. Protein W is not encoded in the photosynthesis gene cluster and our data indicate that only approximately 10% of wild-type Rps. palustris core complexes contain this non-essential subunit; functional and evolutionary consequences of this observation are discussed. The ability to purify uniform RC-LH1 and RC-LH1-protein W preparations will also be beneficial for future structural studies of these bacterial core complexes.

Magnesium chelatase initiates chlorophyll biosynthesis, catalysing the MgATP2−-dependent insertion of a Mg2+ ion into protoporphyrin IX. The catalytic core of this large enzyme complex consists of three subunits: Bch/ChlI, Bch/ChlD and Bch/ChlH (in bacteriochlorophyll and chlorophyll producing species, respectively). The D and I subunits are members of the AAA+ (ATPases associated with various cellular activities) superfamily of enzymes, and they form a complex that binds to H, the site of metal ion insertion. In order to investigate the physical coupling between ChlID and ChlH in vivo and in vitro, ChlD was FLAG-tagged in the cyanobacterium Synechocystis sp. PCC 6803 and co-immunoprecipitation experiments showed interactions with both ChlI and ChlH. Co-production of recombinant ChlD and ChlH in Escherichia coli yielded a ChlDH complex. Quantitative analysis using microscale thermophoresis showed magnesium-dependent binding (Kd 331 ± 58 nM) between ChlD and H. The physical basis for a ChlD–H interaction was investigated using chemical cross-linking coupled with mass spectrometry (XL–MS), together with modifications that either truncate ChlD or modify single residues. We found that the C-terminal integrin I domain of ChlD governs association with ChlH, the Mg2+ dependence of which also mediates the cooperative response of the Synechocystis chelatase to magnesium. The interaction site between the AAA+ motor and the chelatase domain of magnesium chelatase will be essential for understanding how free energy from the hydrolysis of ATP on the AAA+ ChlI subunit is transmitted via the bridging subunit ChlD to the active site on ChlH.

The insertion of magnesium into protoporphyrin initiates the biosynthesis of chlorophyll, the pigment that underpins photosynthesis. This reaction, catalysed by the magnesium chelatase complex, couples ATP hydrolysis by a ChlID motor complex to chelation within the ChlH subunit. We probed the structure and catalytic function of ChlH using a combination of X-ray crystallography, computational modelling, mutagenesis and enzymology. Two linked domains of ChlH in an initially open conformation of ChlH bind protoporphyrin IX and rearrangement of several loops envelops this substrate forming an active site cavity. This induced fit brings an essential glutamate (E660), proposed to be the key catalytic residue for magnesium insertion, into proximity with the porphyrin. A buried solvent channel adjacent to E660 connects the exterior bulk solvent to the active site, forming a possible conduit for delivery of magnesium or abstraction of protons.The central metal ions in the cyclic tetrapyrrole-derived cofactors, magnesium in chlorophyll, cobalt in cobalamin, iron in heme, and nickel in F 430 , are collectively critical for most living systems. These tetrapyrroles underpin photosynthesis, vitamin B12 biosynthesis, respiration and methanogenesis 1 . Despite their apparent simplicity, the insertion of each metal ion into its cognate macrocyclic ring requires surprisingly complex and poorly understood enzymes. These metal ion chelatases can also play a regulatory role in directing and controlling flux down various branches of tetrapyrrole metabolism. A comparatively good structural and mechanistic understanding exists for the relatively simple Class II chelatases 2-6 , but our knowledge of the complex, multisubunit, Class I chelatases, typified here by magnesium chelatase, is more limited.Much of the mechanistic work on this class of chelatases has focused on the reasonably tractable magnesium chelatases (MgCH; E.C.6.6.1.1) from bacteriochlorophyll and chlorophyll producing organisms. MgCH initiates the biosynthetic pathways for these pigments by inserting Mg 2+ into the protoporphyrin macrocycle (Fig. E1) . MgCHs require at least three subunits; chlorophyll producing organisms have ChlI (~35 kDa), ChlD (~75 kDa) and ChlH (~150 kDa), and the closely related proteins from bacteriochlorophyll producing organisms are BchI, BchD, and BchH 7, 8 . In cyanobacteria and higher plants a fourth regulatory subunit, Gun4, is required for full protein activity [9][10][11] .The genes for MgCH were originally identified and recombinant protein expression systems were developed some time ago 7,12 , and extensive kinetic characterization of the MgCH has identified the roles of the subunits [13][14][15][16][17][18] . The current mechanistic and structural data suggest a model for the MgCH mechanism where the two AAA + subunits form the ChlID complex 16,19 . This complex transiently interacts with the body region of the ChlH protein via the C-terminal integrin domain of ChlD 20 , then hydrolyses ATP, which drives a conformational change in the Ch...