NQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain... Show moreNQR is a six-subunit complex that transfers electrons from NADH to ubiquinone, one of the essential enzymes in the bacterial respiratory chain of many pathogens such as Vibrio cholerae, Pseudomonas aeruginosa, Chlamydia trachomatis. Its electron transfer path requires three different flavin cofactors to facilitate: FAD, FMN, and riboflavin. The FMN in subunit B (FMNB) brings electrons to riboflavin and then transfers it to the final electron receptor UQ in subunit B, coupled with the Na+ pumping mechanism. NQR has a unique evolutionary history, and one of the pieces of evidence is that NQR has been reported as the only one flavoenzyme that uses riboflavin as its redox cofactor. However, the binding site of riboflavin has not been well understood. To gain insight into the electron transfer at this site in V.cholerae NQR, we generated mutants at the interface of subunits B, D, and E where the possible location of riboflavin is. To characterize these mutants, we assessed NQR properties with different approaches including enzyme kinetics and flavin radical profiling. We found that the mutagenesis surrounding the hydrophobic pocket disrupted the NQR activity, and cause the loss of neutral radical, but did not interfere with the binding affinity between the substrates and NQR. This study will help to understand electron transfer better in NQR and develop the drugs targeting the riboflavin binding site in the future. Show less
