Calculation of the redox potential of the protein azurin and some mutants

Azurin from Pseudomonas aeruginosa is a small 128-residue, copper-containing protein. Its redox potential can be modified by mutating the protein. Free-energy calculations based on classical molecular-dynamics simulations of the protein and from mutants in aqueous solution at different pH values were used to compute relative redox potentials. The precision of the free-energy calculations with the coupling-parameter approach is evaluated as function of the number and sequence of lambda values, the sampling time and initial conditions. It is found that the precision is critically dependent on the relaxation of hydrogen-bonding networks when changing the atomic-charge distribution due to a change of redox state or pH value. The errors in the free energies range from 1 to 10 k(B)T, depending on the type of process. Only qualitative estimates of the change in redox potential by protein mutation can be obtained.