We were also able to investigate the effects of Y to F mutations in the active site of AcmB on the free energy profile of RHR. Our mutations introduced a minimal disturbance to the protein structure, only removing the tyrosyl OH group. These mutations can be divided into two groups, those targeting the proton relay system (Y115F and Y467F) and those directly interfering with the substrates' activation and enolate stabilization (Y536F, Y363F, Y118F).