Axial interactions in the mixed-valent Cu_a active site and role of the axial methionine in electron transfer

Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent Cu_A sites is less clear. Recently, the mutation of the axial Met to Leu in a Cu_A site engineered into azurin (Cu_A Az) was found to have a limited effect on E⁰ relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on Cu_A Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ΔE ⁰ is attributed to H₂O coordination to the Cu center in the M123L mutant in Cu_A but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in Cu_A and BC (Cu_A ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E⁰ similarly in both. Electron delocalization over two Cu centers in Cu^A was found to minimize the Jahn - Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu-S(Met) bond in oxidized Cu _A is weak (5.2 kcal/mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E⁰ while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions.