Tuning of Enthalpic/Entropic Parameters of a Protein Redox Center through Manipulation of the Electronic Partition Function

Abstract

Manipulation of the partition function (Q) of the redox center Cu from cytochrome c oxidase is attained by tuning the accessibility of a low lying alternative electronic ground state and by perturbation of the electrostatic potential through point mutations, loop engineering and pH variation. We report clear correlations of the entropic and enthalpic contributions to redox potentials with Q and with the identity and hydrophobicity of the weak axial ligand, respectively.

Dramatic Electronic Perturbations of CuA Centers via Subtle Geometric Changes

“…The reduction potential of Tt 3L Met160His at neutral pH is 74 mV, i.e . 187mV lower than Tt CuA, 5 mV lower than Met160His and 168 mV lower than Tt 3L, thus showing the additivity of the effect of first and second sphere mutations on the redox potentials 27 . Protein film voltammetry experiments (Figure S4) yield a reorganization energy for the heterogeneous electron transfer reaction of 0.6 ± 0.1 eV, which is consistent with a π u redox active molecular orbital, as predicted by quantum mechanical calculations 7,13 and experimentally verified for the other Cu A variants.…”

“…For the calculations we corrected by relative molar absorptivity and by the contribution of the overlapping LMCT N His→cu band. 27 Thus, the probability of populating the π u ground-state (P π ) for the different variants at different conditions were calculated as: Pπ=A27000−0,32.A21000A27000+A21000…”

CuA-based chimeric T1 copper sites allow for independent modulation of reorganization energy and reduction potential

“…5D ), which can be rationalized in terms of destabilization of Cu 2+ relative to Cu 1+ by increasingly hydrophobic environments. 13 , 17 , 28 , 77 – 79 Note that 2R2R– Tt –Cu A is the only variant that strongly deviates from all the correlations shown in Fig. 5 and has by far the highest E °′ of the series (540 mV; Table S1 † ).…”

“…1,4 Therefore, along with point mutations, replacement of the entire ligand loop by sequences from other proteins or unnatural sequences, 5 has become one of the preferred strategies for modulating the electronic properties of T1 4,[6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] and Cu A sites. [24][25][26][27][28][29][30] This methodology has also allowed for the successful insertion of Cu A sites into the scaffold of T1 [31][32][33][34][35] proteins and vice versa. 36 So far, most efforts have focused on tuning reduction potentials (E 0 ) of T1-like mononuclear centers through rst and second sphere perturbations, attaining up to 700 mV modulation.…”

Modulation of Functional Features in Electron Transferring Metalloproteins

“…A at the level of the three loops that surround the metal site (see Figure 6) As modification of the strong Cys and His ligands of the Cu A site are known to be disruptive, first coordination sphere modifications were restricted to the replacement of the weak axial ligand Met160 by a variety of amino acids. 3,[48][49][50][51][52][53][54] Second sphere modifications, on the other hand, were accomplished by replacement of either one or the three loops that surround the metal site (Figures 6 and Table 1) by the homologous sequences from Homo sapiens and Arabidopsis thaliana with preservation of the conserved ligand set. 50,54 Moreover, combined first and second coordination sphere modification was achieved by introducing the Met160His point mutation in the so-called Tt-3L-Hs chimera.…”