High-pressure tuning of primary photochemistry in bacterial photosynthesis: membrane-bound versus detergent-isolated reaction centers

Abstract

While photosynthesis thrives at close to normal pressures and temperatures, it is presently well known that life is similarly commonplace in the hostile environments of the deep seas as well as around hydrothermal vents. It is thus imperative to understand how key biological processes perform under extreme conditions of high pressures and temperatures. Herein, comparative steady-state and picosecond time-resolved spectroscopic studies were performed on membrane-bound and detergent-purified forms of a YM210W mutant reaction center (RC) from Rhodobacter sphaeroides under modulating conditions of high hydrostatic pressure applied at ambient temperature. A previously established breakage of the lone hydrogen bond formed between the RC primary donor and the protein scaffold was shown to take place in the membrane-bound RC at an almost 3 kbar higher pressure than in the purified RC, confirming the stabilizing role of the lipid environment for membrane proteins. The main change in the multiexponential decay of excited primary donor emission across the experimental 10 kbar pressure range involved an over two-fold continuous acceleration, the kinetics becoming increasingly mono-exponential. The fastest component of the emission decay, thought to be largely governed by the rate of primary charge separation, was distinctly slower in the membrane-bound RC than in the purified RC. The change in character of the emission decay with pressure was explained by the contribution of charge recombination to emission decreasing with pressure as a result of an increasing free energy gap between the charge-separated and excited primary donor states.Finally, it was demonstrated that, in contrast to a long-term experimental paradigm, adding a combination of sodium ascorbate and phenazine methosulfate to the protein solution potentially distorts natural photochemistry in bacterial RCs.

Effects of High Hydrostatic Pressure on the Spectral and Temporal Characteristics of Fluorescence of Photosystem II Core Complex of Thermosthicus Vulcanus

“…For instance, at 300 K, the 870-nm absorption band of the primary donor in the RC of Rhodobacter sphaeroides was found to redshift at a rate of -68 cm ¹ / kbar (Clayton and Devault 1972). The observed weakening of the special pair uorescence in compressed bRCs at room temperature (Timpmann et al 2020) was attributed to an increased free energy gap between the RC excited state and the charge-separated state, reducing the contribution of charge recombination to the overall emission and modifying the uorescence kinetics. Similar large slopes have been noted in cyclic bacterial light-harvesting complexes LH2 and LH1 (Freiberg et al 1993) (Tars et al 1994), where bacteriochlorophyll pigments are arranged in strongly excitonically coupled dimers.…”

Hydrostatic High-Pressure-Induced Denaturation of LH2 Membrane Proteins

“…Despite the apparent full coverage by detergent molecules of transmembrane hydrophobic parts of the polypeptides, the protection attained by native membrane lipids is more complete. In high-pressure measurements, this is evidenced by significantly higher denaturation pressures demonstrated for native membrane-embedded complexes than for detergent-isolated complexes. , …”

“…In high-pressure measurements, this is evidenced by significantly higher denaturation pressures demonstrated for native membrane-embedded complexes than for detergentisolated complexes. 30,62 As is obvious from Figures 6 and 7, the Trp residues effectively guarding the entrances to the hydrophilic core of the protein are not positioned in identical locations and are not equally accessible to the aqueous environment. Therefore, in principle, the protein fluorescence spectrum is expected to be a heterogeneous mixture of contributions from individual Trp residues rather than a sum of more-or-less identical homogeneously broadened spectral components.…”

Harnessing Pressure-Axis Experiments to Explore Volume Fluctuations, Conformational Substates, and Solvation of Biomolecular Systems

“…Compared to folding studies, work on the fast kinetics of protein-related reactions, such as photoinduced biochemical processes, is still scarce. − Klink et al studied the pressure dependence of the photocycle kinetics of bacteriorhodopsin from Halobacterium salinarium, a light-driven proton pump, at pressures up to 4 kbar and modeled the kinetics by rate constants in the millisecond-to-microsecond time range of nine kinetically distinguishable states consisting of specific spectral components. From the pressure dependence, the volume differences between these spectral states could be determined and a model could be developed to explain the magnitude and sign of the volume changes at the various kinetic transitions …”