The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Q site of bacterial cytochrome bc.

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron‑sulfur protein (ISP) with its FeS cluster, cyt c and cyt b, forming two catalytic domains, the Q (hydroquinone (QH) oxidation) and Q (quinone (Q) reduction) sites. At the Q site, the electron transfer pathways originating from QH oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the FeS cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH oxidation at the Q site of cyt bc.

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