X-Ray Crystal and Cryo-Electron Microscopy Structure Analysis Unravels How the Unique Thylakoid Lipid Composition Is Utilized by Cytochrome b6f for Driving Reversible Proteins’ Reorganization During State Transitions

The rapid regulatory mechanism of light-induced state transitions (STs) in oxygenic photosynthesis is particularly appealing for membrane-based applications. This interest stems from the unique ability of the thylakoid membrane protein cytochrome <i>b</i>₆<i>f</i> (cyt<i>b</i>₆<i>f</i>) to increase or decrease its hydrophobic thickness (d_P) in parallel with the reduction or oxidation of the PQ pool induced by changes in light quality. This property appears to be the long-sought biophysical driver behind the reorganizations of membrane proteins during STs. This study decisively advances the hydrophobic mismatch (HMM) model for cyt<i>b</i>₆<i>f</i>-driven STs by thoroughly analyzing thirteen X-ray crystal and eight cryo-electron microscopy cyt<i>b</i>₆<i>f</i> structures. It uncovers the lipid nanoenvironments that cyt<i>b</i>₆<i>f</i>, with different hydrophobic thicknesses, selectively attracts. Under optimal, stationary conditions for photosynthesis in low light, when there is hydrophobic matching between the hydrophobic thicknesses of cyt<i>b</i>₆<i>f</i> d_P and that of the bulk thylakoid lipid phase d_L, d_P = d_L, cyt<i>b</i>₆<i>f</i> predominantly binds to anionic lipids-several phosphatidylglycerol (PG) molecules and one sulfoquinovosyldiacylglycerol (SQDG) molecule. Upon the induction of the transition to State 2, when d_P increases and induces a positive HMM (d_P > d_L), the neutral, non-bilayer-forming lipid monogalactosyldiacylglycerol (MGDG) replaces some of the bound PGs. Upon the induction of the transition to State 1, when d_P decreases and induces a negative HMM (d_P < d_L), PGs and SQDG detach from their binding sites, and two neutral, bilayer-forming lipids such as digalactosyldiacylglycerol (DGDG) occupy two sites. Additionally, this research uncovers two lipid-mediated signaling pathways from Chl<i>a</i> to the center of flexibility, the Phe/Tyr124<i>^fg</i>^-loop-suIV residue-one of which involves β-carotene. This study identifies two novel types of lipid raft-like nanodomains that are devoid of typical components, such as sphingomyelin and cholesterol. These findings firmly validate the HMM model and underscore the STs as the first recognized functional process that fully utilizes the unique and evolutionarily conserved composition of just four thylakoid lipid classes. This research contributes to our understanding of membrane dynamics in general and STs in particular. It introduces a novel and simple approach for reversible protein reorganization driven purely by biophysical mechanisms, with promising implications for various membrane-based applications.