Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs’ functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains.

F-type ATP synthases play a key role in oxidative and photophosphorylation processes generating adenosine triphosphate (ATP) for most biochemical reactions in living organisms. In contrast to the mitochondrial F_OF₁-ATP synthases, those of chloroplasts are known to be mostly monomers with approx. 15% fraction of oligomers interacting presumably non-specifically in a thylakoid membrane. To shed light on the nature of this difference we studied interactions of the chloroplast ATP synthases using small-angle X-ray scattering (SAXS) method. Here, we report evidence of I-shaped dimerization of solubilized F_OF₁-ATP synthases from spinach chloroplasts at different ionic strengths. The structural data were obtained by SAXS and demonstrated dimerization in response to ionic strength. The best model describing SAXS data was two ATP-synthases connected through F₁/F₁′ parts, presumably via their δ-subunits, forming “I” shape dimers. Such I-shaped dimers might possibly connect the neighboring lamellae in thylakoid stacks assuming that the F_OF₁ monomers comprising such dimers are embedded in parallel opposing stacked thylakoid membrane areas. If this type of dimerization exists in nature, it might be one of the pathways of inhibition of chloroplast F_OF₁-ATP synthase for preventing ATP hydrolysis in the dark, when ionic strength in plant chloroplasts is rising. Together with a redox switch inserted into a γ-subunit of chloroplast F_OF₁ and lateral oligomerization, an I-shaped dimerization might comprise a subtle regulatory process of ATP synthesis and stabilize the structure of thylakoid stacks in chloroplasts.

We present the results of the study of the structural distinctions between the purple membrane (PM) of Halobacterium salinarum and the claret membrane (CM) of Haloquadratum walsbyi using small-angle and wide-angle X-ray scattering. PM and CM are rhodopsin-rich membrane regions. They are crucial for light-driven energy processes in extreme halophilic archaea. While PM structure has been extensively characterized, the structure of CM remains less understood. According to our data, PM exhibits a well-defined hexagonal crystalline lattice with unit cell parameters of a = b ≈ 62 Å, which is consistent with published data. Conversely, CM showed unexpected diffraction patterns, best fitted by rhombohedral lattice with unit cell dimensions a = b = 27.9 Å; γ = 82.9°. But the presence of several unindexed peaks indicates the complexity of the CM sample. These structural differences are attributed to variations in native lipid and pigment compositions, specifically the presence of bacterioruberin in CM, as confirmed by UV-visible spectroscopy. Bacterioruberin, a hallmark of CM, contributes to its red-shifted absorption spectrum, indicative of its organized state within the membrane. However, denaturing 12% polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopy reveal potential contamination in CM samples, suggesting the need for improved purification protocols. The findings highlight significant structural disparities between PM and CM, with implications for understanding membrane organization. This research underscores the critical role of lipids and pigments in shaping the supramolecular architecture of archaeal membranes and provides a foundation for future studies into their functional adaptations.