Cryo-EM structure of HQNO-bound Alternative Complex III from the anoxygenic phototrophic bacterium Chloroflexus aurantiacus.

Alternative complex III (ACIII) couples quinol oxidation and electron acceptor reduction with potential transmembrane proton translocation. It is compositionally and structurally different from the cytochrome bc1/b6f complexes, but functionally replaces these enzymes in the photosynthetic and/or respiratory electron transport chains (ETCs) of many bacteria. However, the true compositions and architectures of ACIIIs remain unclear, as do their structural and functional relevance in mediating the ETCs. We here determined cryogenic electron microscopy structures of photosynthetic ACIII isolated from Chloroflexus aurantiacus (CaACIIIp), in apo-form and in complexed form bound to a menadiol analog 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Besides six canonical subunits (ActABCDEF), the structures revealed conformations of two previously unresolved subunits, ActG and I, which contributed to the complex stability. We also elucidated the structural basis of menaquinol oxidation and subsequent electron transfer along the [3Fe-4S]-6 hemes wire to its periplasmic electron acceptors, using electron paramagnetic resonance (EPR), spectroelectrochemistry, enzymatic analyses and molecular dynamics (MD) simulations. A unique insertion loop in ActE was shown to function in determining the binding specificity of CaACIIIp for downstream electron acceptors. This study broadens our understanding of the structural diversity and molecular evolution of ACIIIs, enabling further investigation of the (mena)quinol oxidoreductases evolved coupling mechanism in bacterial energy conservation.

A cytochrome c551 mediates the cyclic electron transport chain of the anoxygenic phototrophic bacterium Roseiflexus castenholzii.

Roseiflexus castenholzii is a gram-negative filamentous phototrophic bacterium that carries out anoxygenic photosynthesis through a cyclic electron transport chain (ETC). The ETC is composed of a reaction center (RC)-light-harvesting (LH) complex (rcRC-LH); an alternative complex III (rcACIII), which functionally replaces the cytochrome bc1/b6f complex; and the periplasmic electron acceptor auracyanin (rcAc). Although compositionally and structurally different from the bc1/b6f complex, rcACIII plays similar essential roles in oxidizing menaquinol and transferring electrons to the rcAc. However, rcACIII-mediated electron transfer (which includes both an intraprotein route and a downstream route) has not been clearly elucidated, nor have the details of cyclic ETC. Here, we identify a previously unknown monoheme cytochrome c (cyt c551) as a novel periplasmic electron acceptor of rcACIII. It reduces the light-excited rcRC-LH to complete a cyclic ETC. We also reveal the molecular mechanisms involved in the ETC using electron paramagnetic resonance (EPR), spectroelectrochemistry, and enzymatic and structural analyses. We find that electrons released from rcACIII-oxidized menaquinol are transferred to two alternative periplasmic electron acceptors (rcAc and cyt c551), which eventually reduce the rcRC to form the complete cyclic ETC. This work serves as a foundation for further studies of ACIII-mediated electron transfer in anoxygenic photosynthesis and broadens our understanding of the diversity and molecular evolution of prokaryotic ETCs.

Carotenoid assembly regulates quinone diffusion and the Roseiflexus castenholzii reaction center-light harvesting complex architecture.

Carotenoid (Car) pigments perform central roles in photosynthesis-related light harvesting (LH), photoprotection, and assembly of functional pigment-protein complexes. However, the relationships between Car depletion in the LH, assembly of the prokaryotic reaction center (RC)-LH complex, and quinone exchange are not fully understood. Here, we analyzed native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. Newly identified exterior Cars functioned with the bacteriochlorophyll B800 to block the proposed quinone channel between LHαß subunits in the nRC-LH, forming a sealed LH ring that was disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling. dRC-LH lacked subunit X, leading to an exposed LH ring with a larger opening, which together accelerated the quinone exchange rate. We also assigned amino acid sequences of subunit X and two hypothetical proteins Y and Z that functioned in forming the quinone channel and stabilizing the RC-LH interactions. This study reveals the structural basis by which Cars assembly regulates the architecture and quinone exchange of bacterial RC-LH complexes. These findings mark an important step forward in understanding the evolution and diversity of prokaryotic photosynthetic apparatus.

Photosynthesis is a biological process that converts energy from sunlight into a form of chemical energy that supports almost all life on Earth. Over the course of evolution, photosynthesis has gone from being only performed by bacteria to appearing in algae and green plants. While this has given rise to a range of different machineries for photosynthesis, the process always begins the same way: with a structure called the reaction center-light harvesting (RC-LH) complex. Two pigments in the light-harvesting (LH) region ­ known as chlorophyll and carotenoids ­ absorb light energy and transfer it to another part of the complex known as the quinone-type reaction center (RC). This results in the release of electrons that interact with a molecule called quinone converting it to hydroquinone. The electron-bound hydroquinone then shuttles to other locations in the cell where it initiates further steps that ultimately synthesize forms of chemical energy that can power essential cellular processes. In photosynthetic bacteria, hydroquinone must first pass through a ring structure in the light harvesting region in order to leave the reaction center. Previous studies suggest that carotenoids influence the architecture of this ring, but it remains unclear how this may affect the ability of hydroquinone to move out of the RC-LH complex. To investigate, Xin, Shi, Zhang et al. used a technique called cryo-electron microscopy to study the three-dimensional structure of RC-LH complexes in one of the first bacterial species to employ photosynthesis, Roseiflexus castenholzii. The experiments found that fully assembled complexes bind two groups of carotenoids: one nestled in the interior of the LH ring and the other on the exterior. The exterior carotenoids work together with bacteriochlorophyll molecules to form a closed ring that blocks hydroquinone from leaving the RC-LH complex. To allow hydroquinone to leave, two groups of regulatory proteins, including a cytochrome and subunit X, then disrupt the structure of the ring to 'open' it up. These findings broaden our knowledge of the molecules involved in photosynthesis. A better understanding of this process may aid the development of solar panels and other devices that use RC-LH complexes rather than silicon or other inorganic materials to convert energy from sunlight into electricity.

Structural basis of a bi-functional malonyl-CoA reductase (MCR) from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii.

Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO2 fixation cycles of Chloroflexaceae green non-sulfur bacteria and the archaea Crenarchaeota. However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium Roseiflexus castenholzii (RfxMCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP+ and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length RfxMCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP+-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.

Preparation of Photo-Bioelectrochemical Cells With the RC-LH Complex From Roseiflexus castenholzii.

Roseiflexus castenholzii is an ancient green non-sulfur bacteria that absorbs the solar energy through bacteriochlorophylls (BChls) bound in the only light harvesting (LH) complex, and transfers to the reaction center (RC), wherein primary charge separation occurs and transforms the energy into electrochemical potentials. In contrast to purple bacteria, R. castenholzii RC-LH (rcRC-LH) does not contain an H subunit. Instead, a tightly bound tetraheme cytochrome c subunit is exposed on the P-side of the RC, which contains three BChls, three bacteriopheophytins (BPheos), two menaquinones, and one iron for electron transfer. These novel structural features of the rcRC-LH are advantageous for enhancing the electron transfer efficiency and subsequent photo-oxidation of the c-type hemes. However, the photochemical properties of rcRC-LH and its applications in developing the photo-bioelectrochemical cells (PBECs) have not been characterized. Here, we prepared a PBEC using overlapped fluorine-doped tin oxide (FTO) glass and Pt-coated glass as electrodes, and rcRC-LH mixed with varying mediators as the electrolyte. Absence of the H subunit allows rcRC-LH to be selectively adhered onto the hydrophilic surface of the front electrode with its Q-side. Upon illumination, the photogenerated electrons directly enter the front electrode and transfer to the counter electrode, wherein the accepted electrons pass through the exposed c-type hemes to reduce the excited P+, generating a steady-state current of up to 320 nA/cm2 when using 1-Methoxy-5-methylphenazinium methyl sulfate (PMS) as mediator. This study demonstrated the novel photoelectric properties of rcRC-LH and its advantages in preparing effective PBECs, showcasing a potential of this complex in developing new type PBECs.

Structural basis of allosteric regulation of Tel1/ATM kinase.

ATM/Tel1 is an apical kinase that orchestrates the multifaceted DNA damage response. Mutations of ATM/Tel1 are associated with ataxia telangiectasia syndrome. Here, we report cryo-EM structures of symmetric dimer (4.1 Å) and asymmetric dimer (4.3 Å) of Saccharomyces cerevisiae Tel1. In the symmetric state, the side chains in Tel1 C-terminus (residues 1129-2787) are discernible and an atomic model is built. The substrate binding groove is completely embedded in the symmetric dimer by the intramolecular PRD and intermolecular LID domains. Point mutations in these domains sensitize the S. cerevisiae cells to DNA damage agents and hinder Tel1 activation due to reduced binding affinity for its activator Xrs2/Nbs1. In the asymmetric state, one monomer becomes more compact in two ways: the kinase N-lobe moves down and the Spiral of α-solenoid moves upwards, which resemble the conformational changes observed in active mTOR. The accessibility of the activation loop correlates with the synergistic conformational disorders in the TRD1-TRD2 linker, FATC and PRD domains, where critical post-translational modifications and activating mutations are coincidently condensed. This study reveals a tunable allosteric network in ATM/Tel1, which is important for substrate recognition, recruitment and efficient phosphorylation.

3.9 Å structure of the yeast Mec1-Ddc2 complex, a homolog of human ATR-ATRIP.

The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master regulator of DNA damage response and replication stress in humans, but the mechanism of its activation remains unclear. ATR acts together with its partner ATRIP. Using cryo-electron microscopy, we determined the structure of intact Mec1-Ddc2 (the yeast homolog of ATR-ATRIP), which is poised for catalysis, at a resolution of 3.9 angstroms. Mec1-Ddc2 forms a dimer of heterodimers through the PRD and FAT domains of Mec1 and the coiled-coil domain of Ddc2. The PRD and Bridge domains in Mec1 constitute critical regulatory sites. The activation loop of Mec1 is inhibited by the PRD, revealing an allosteric mechanism of kinase activation. Our study clarifies the architecture of ATR-ATRIP and provides a structural framework for the understanding of ATR regulation.