Cellular localization of cytochrome bd in cyanobacteria using genetic code expansion

Photosynthesis is one of the most fundamental and complex mechanisms in nature. It is a well‐studied process, however, some photosynthetic mechanisms are yet to be deciphered. One of the many proteins that take part in photosynthesis, cytochrome bd, is a terminal oxidase protein that plays a role both in photosynthesis and in respiration in various organisms, specifically, in cyanobacteria. To clarify the role of cytochrome bd in cyanobacteria, a system for the incorporation of an unnatural amino acid into a genomic membrane protein cytochrome bd was constructed in Synechococcus sp. PCC7942. N‐propargyl‐ l‐lysine (PrK) was incorporated into mutants of cytochrome bd. Incorporation was verified and the functionality of the mutant cytochrome bd was tested, revealing that both electrochemical and biochemical activities were relatively similar to those of the wild‐type protein. The incorporation of PrK was followed by a highly specific labeling and localization of the protein. PrK that was incorporated into the protein enabled a “click” reaction in a bio‐orthogonal manner through its alkyne group in a highly specific manner. Cytochrome bd was found to be localized mostly in thylakoid membranes, as was confirmed by an enzyme‐linked immunosorbent assay, indicating that our developed localization method is reliable and can be further used to label endogenous proteins in cyanobacteria. A localization method that can be used to label endogenous proteins in cyanobacteria was established. We demonstrated this method utilizing a bio‐orthogonal and highly specific ‘click’ reaction between a small fluorescent linker and an unnatural amino acid that was incorporated into a membrane protein (cytochrome bd), using a system that introduces unnatural amino acid in a site‐specific manner in cyanobacteria. By detecting the fluorescent signal of the linker, cytochrome bd was found to be localized mostly in thylakoid membranes.