Probing the local lipid environment of the cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Abstract

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism

“…Cytbc1 SMALPs and Rba. sphaeroides chromatophores were prepared and analyzed by thin layer chromatography (TLC) and phosphate quantification as previously described (Swainsbury et al, 2018), with the exception that lipid band intensities were plotted as a fraction of the total.…”

Membrane protein nanoparticles: the shape of things to come

“…Lipids have been detected in SMALPs in several studies and lipid preferences for many proteins have been characterised for the first time using SMALPs [32,33]. A study of Rhodobacter sphaeroides proteins solubilised by SMA showed that they retained significant numbers of lipids [34]. However, they also related the solubilisation efficiency of R. sphaeroides MPs to their local lipid environment.…”

Engineering of B800 bacteriochlorophyll binding site specificity in the Rhodobacter sphaeroides LH2 antenna

“…Light is absorbed by either the LH2 or LH1 antenna, arranged in protein-rich arrays, and excitation energy is transferred within picoseconds to the RC, where charge separation drives the reduction of quinone to quinol and the oxidation of cytochrome c 2 [ [5] , [6] , [7] , [8] , [9] , [10] ] . These products are utilised by the cytochrome bc 1 complex, which resides in a locally lipid-rich region, to re-reduce oxidised cytochrome c 2 and oxidise quinol to quinone [ 3 , 4 , 11 , 12 ]. This cyclic process results in the transport of protons into the lumen (periplasm) of the chromatophore, which drive ATP generation by ATP synthase [ 3 , 4 ].…”