An Attempt to Increase Thermostability of the Mutant Photosynthetic Reaction Center of Cereibacter sphaeroides Using Disulfide Bonds

Abstract

Methods of site-directed mutagenesis are successfully used in structural and functional studies of photosynthetic reaction centers (RCs). It has been noted that many mutations near electron transfer cofactors reduce temperature stability of the Cereibacter sphaeroides RCs and affect amount of RCs in the membranes. We previously reported [Selikhanov et al. (2023) Membranes, 25, 154] that introduction of inter-subunit disulfide bridges on the periplasmic or cytoplasmic surface of the complex promotes increase in thermal stability of the C. sphaeroides RCs. In this work, an attempt was made to increase thermal stability of the mutant RC with the Ile M206 – Gln substitution by introducing inter-subunit disulfide bonds. This RC is of considerable interest for studying mechanisms of early electron transfer processes in RCs. The effect of mutations on the amount of RCs in chromatophores was analyzed and it was found that the I(M206)Q mutation leads to twofold decrease in the RC content in chromatophores, introduction of disulfide bonds on the cytoplasmic or periplasmic sides of the complex reduces the amount of RCs in membranes by one third, the triple substitution I(M206)Q/G(M19)C/T(L214)C reduces the amount of RCs in membranes almost 4-fold, and the substitutions I(M206)Q/V(M84)C/G(L278)C lead to disruption of RC assembly in the membrane. It was shown that introduction of the inter-subunit S-S bond on the cytoplasmic surface of the complex did not have a significant effect on thermal stability of the I(M206)Q RC. Our own and literature data on the factors influencing assembly processes and ensuring stability of the structure of integral membrane complexes are discussed.