Abstract
Bacteriorhodopsin (BR) functions as a light-driven proton pump that transitions between different states during the photocycle, such as all-trans (AT; BR568) and 13-cis, 15-syn (CS; BR548) state and K, L, M1, M2, N, and O intermediates. In this study, we used in situ photoirradiation 13C solid-state NMR to observe a variety of photo-intermediates and photoreaction pathways in [20-13C]retinal-WT-BR and its mutant [20-13C, 14-13C]retinal-D96N-BR. In WT-BR, the CS state converted to the CS* intermediate under photoirradiation with green light at −20 °C and consequently converted to the AT state in the dark. The AT state converted to the N intermediate under irradiation with green light. In D96N-BR, the CS state was converted to the CS* intermediate at −30 °C and consequently converted to the AT state. Simultaneously, the AT state converted to the M and L intermediates under green light illumination at −30 °C and subsequently converted to the AT state in the dark. The M intermediate was directly excited to the AT state by UV light illumination. We demonstrated that short-lived photo-intermediates could be observed in a stationary state using in situ photoirradiation solid-state NMR spectroscopy for WT-BR and D96N-BR, enabling insight into the light-driven proton pump activity of BR.
Highlights
Accepted: 26 February 2022Bacteriorhodopsin (BR) is a retinal-containing transmembrane H+ pump found in the purple membrane (PM) of the archaeon Halobacterium salinarum, where it generates a proton gradient across the cytoplasmic membrane for ATP synthesis [1–3]
High-resolution solid-state 13C and 15N NMR spectra were recorded by cross polarizationmagic angle spinning (CP-MAS) on a Chemagnetics CMX-400 Infinity FT-NMR spectrometer operated at 100.1 MHz for 13 C, 40.3 MHz for 15 N and 398.1 MHz for 1 H
The 13 C CP/MAS NMR signals of [20-13 C]retinal-BR and [20-13 C, 14-13 C]retinal-D96NBR were measured under green light illumination
Summary
Bacteriorhodopsin (BR) is a retinal-containing transmembrane H+ pump found in the purple membrane (PM) of the archaeon Halobacterium salinarum, where it generates a proton gradient across the cytoplasmic membrane for ATP synthesis [1–3]. The 26 kDa BR protein contains seven transmembrane α-helices and a retinal group covalently bonded to the ε-amino group of Lys216 in helix G. An extensive threedimensional hydrogen-bonded network of protein residues and seven water molecules connects the buried retinal Schiff base (SB) and the proton acceptor Asp to the membrane surface [4]. These structural changes suggest that protonation of Asp initiates a cascade of atomic displacements in the extracellular region, causing the release of a proton to the surface (see Figure 1A) [5,6]
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