In aqueous solution dissolved inorganic carbon (DIC) is in a constant pH-dependent equilibrium between CO2 and bicarbonate. For photosynthetic organisms in the aqueous environment, such as cyanobacteria and green algae, carbon uptake is often a growth-limiting factor. In order to survive under low CO2 conditions and to mitigate photorespiration, these organisms have evolved active and facilitated uptake systems which as a whole build up the highly effective carbon-concentrating mechanisms (CCM) to enhance photosynthetic CO2 fixation. At acidic to neutral conditions, most of the DIC exists as dissolved CO2, whereas under alkaline conditions bicarbonate is the main inorganic carbon source. Both show very different membrane permeability: HOC3- is ionized and requires transporter proteins, while CO2 has no charge and is thus able to diffuse through membranes where it is rapidly converted to bicarbonate and trapped in the cell using membrane bound carbonic anhydrases.
Our lab is interested in studying the structure and mechanism of the specific transporters and uptake machineries in the cyanobacterial cell. Mechanistic insights might be implemented in generating improved industrial microbes for the production of chemical compounds, creating solar fuels, and sequestering CO22 to counteract global warming. The lab is currently funded by an Emmy Noether grant from the DFG.
The cyanobacterial CCM takes advantage of the functional and structural diversity of the cyanobacterial photosynthetic NADH dehydrogenase-like complex I (NDH-1). Specialized forms of the NDH-1 complex, the NDH-1MS and NDH-1MS’ complexes, contain alternative subunits that drive the conversion of CO2 to HCO3- against the equilibrium using redox energy supplied by ferredoxin (Fd). We are interested in studying the mechanism of the cyanobacterial photosynthetic complex I and could recently solve the structure of the NDH-1L and NDH-1MS complexes using high-resolution cryoEM single particle analysis.
Structure of the carbon concentrating photosynthetic complex I (NDH-1MS)
Our structure of the NDH-1MS complex shows how modular adaptations enabled the cyanobacterial photosynthetic complex I to concentrate CO2 using a redox-driven proton pumping machinery (Schuller et al. NatureComm., 2020). On the cytoplasmic side of the protein pumping arm, the carbonic anhydrase binds CupA and the small protein CupS, giving the complex its U-shaped architecture. The subunit of the carbonic anhydrase (CA) has a fold that is different from any other CA described so far. Even more surprisingly CupA also possess a unique catalytic site architecture and coordination of the catalytic Zn2+ ion. It is coordinated by His130 and by deprotonated Arg135.
Another unexpected feature of this remarkable machine is that the "proton-pumping" subunit NdF3 lacks the charged residues that are relevant for the formation of a proton line, but instead has a hydrophobic gas channel leading to the active CupA site. Both observations, the absence of proton pumping and the transport of CO2 through the gas tunnel, are supported by MD simulations. In contrast, the directly adjacent antiporter-like subunit NdhD3 is a functional proton pump. We propose that the carbon-concentrating photosynthetic complex I uses a bimodal "push and pull" mechanism to shift the CO2 hydration equilibrium. Two components drive the vectorial CO2 hydration: the pumping of CO2 into NdhF3, which produces a high local concentration, and the active removal of H+ by NdhD3.
The carbon concentrating photosynthetic complex I remains a current focus of the lab. We have two major interests: (1) We want to understand the enzymatic mechanism of CupA. (2) We study the long-range communication between electron transduction, the proton pumping machinery, and the carbonic anhydrase activity of the complex. We are using a unique and powerful combination of structural biology, functional studies and MD simulations.
Structure of the photosynthetic complex I (NDH-1L)
Our near-atomic model of the NDH-1L complex sheds light on the divergent evolution of respiratory and photosynthetic complex I (Schuller et al. Science, 2019). The two complexes differ substantially in the structure of the peripheral arm (Q-module), which is responsible for electron transfer via iron-sulfur centers towards the quinone-based terminal acceptor; yet, they share high similarity in the membrane-embedded antiporter-like region (P-module). Most of the eight-additional photosynthesis-specific subunits are located in the Q-module, entwined with each other via novel extensions. The structure enabled us to propose a putative Fd binding site on the top of Q-module of NDH-1L, with the NdhS subunit as one of the central elements. To further pinpoint this interaction, we performed NMR chemical shift perturbation analyses with recombinant NdhS. This identified the flexible C-terminus of the isolated NdhS as the major interaction site with Fd, suggesting that Fd may be efficiently recruited through a ‘fly-casting’ interaction mode.