A few key residues determine the high redox potential shift in azurin mutants

Abstract

The wide range of variability of the reduction potential (E(0)) of blue-copper proteins has been the subject of a large number of studies in the past several years. In particular, a series of azurin mutants have been recently rationally designed tuning E(0) over a very broad range (700 mV) without significantly altering the redox-active site [Marshall et al., Nature, 2009, 462, 113]. This clearly suggests that interactions outside the primary coordination sphere are relevant to determine E(0) in cupredoxins. However, the molecular determinants of the redox potential variability are still undisclosed. Here, by means of atomistic molecular dynamics simulations and hybrid quantum/classical calculations, the mechanisms that determine the E(0) shift of two azurin mutants with high potential shifts are unravelled. The reduction potentials of native azurin and of the mutants are calculated obtaining results in good agreement with the experiments. The analysis of the simulations reveals that only a small number of residues (including non-mutated ones) are relevant in determining the experimentally observed E(0) variation via site-specific, but diverse, mechanisms. These findings open the path to the rational design of new azurin mutants with different E(0).

Prediction of Reduction Potentials of Copper Proteins with Continuum Electrostatics and Density Functional Theory

“…Hence, the effect of increased flexibility of the protein due to reorganization of the hydrogen bonds can be ruled out. Another study on a double mutant that contained the N47S replacement suggested that an increased reduction potential results from interactions between the two ligand‐containing loops …”

“…Clearly, DFT has an advantage over continuum electrostatics for this situation and approaches the experimental reduction potential change better. The effect of hydrogen bond formation by Asn 114 in the F114N mutant was previously reported, and found to switch between the backbone amide of His 117 and either the amide of Gly 45 or carbonyl of Val 43 . The formation of the latter interactions was found to be a key determinant of the reduction potential.…”

“…Another example is the F114N mutant (Scheme ), in which a hydrogen bond partner is introduced near to the Gly 45 and His 117 amino acids. A recent computational study suggested that hydrogen bonds are formed between the sidechain of Asn 114 and the backbone amide of His 117 and the carbonyl group of Gly 45 . The formation of such a hydrogen bond could perturb the coordination geometry, leading to a change in reduction potential which cannot be captured by continuum electrostatics.…”

“…Adding additional positive charge in this position (D11K), however, does not result in a much higher reduction potential (+96 mV). Our observation for the D11K mutant is in agreement with a recent quantum mechanics/molecular mechanics study on azurin and a few mutants . Also interesting is that the average distance between the mutated residue and the copper for some of these mutants is relatively large yet the calculations suggest reasonable shifts in reduction potential.…”

“…Methods which incorporate protein dynamics have been shown to compute the reduction potentials of some copper protein mutants with good agreement with experiment ,. Unfortunately, these methods incur a significant computational cost especially because they need to sample a significant number of protein conformations, which limit the number of mutants that can be investigated.…”