Dramatic Electronic Perturbations of Cu_A Centers via Subtle Geometric Changes

Abstract

CuA is a binuclear copper site acting as electron entry port in terminal heme-copper oxidases. In the oxidized form, CuA is a mixed valence pair whose electronic structure can be described using a potential energy surface with two minima: σu* and πu, that are variably populated at room temperature. We report that mutations in the first and second coordination spheres of the binuclear metallocofactor can be combined in an additive manner to tune the energy gap and, thus, the relative populations of the two lowest-lying states. A series of designed mutants span σu*/πu energy gaps ranging from 900 to 13 cm−1. The smallest gap corresponds to a variant with an effectively degenerate ground state. All engineered sites preserve the mixed-valence character of this metal center and the electron transfer functionality. An increase of the Cu-Cu distance less than 0.06 Å modifies the σu*/πu energy gap by almost two orders of magnitude, with longer distances eliciting a larger population of the πu state. This scenario offers a stark contrast to synthetic systems, as model compounds require a lengthening of 0.5 Å in the Cu-Cu distance to stabilize the πu state. These findings show that the tight control of the protein environment allows drastic perturbations in the electronic structure of CuA sites with minor geometric changes.

Mechanistic basis of substrate–O2coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study

“…Overall, these results show that the mechanism of substrate–O 2 coupling can be effected through rearrangement of the copper coordination geometry and subsequent changes in the d-orbital electronics, with minimal change in the rest of the protein backbone structure. These results underline recent observations in other copper proteins ( 60 ) that minimal structural changes can be coupled to large electronic changes at the copper active site.…”

Modulation of Functional Features in Electron Transferring Metalloproteins

“…Interestingly, the introduction of the Met160His mutation into the Tt-3L-Hs chimera to produce the Tt-3L-M160H variant results in a downshift of E 0 with respect to WT Tt-Cu A , which approximately corresponds to the sum of the downshifts induced by the individual first and second sphere modifications. 53 As discussed in the preceding section, inner and outer sphere mutations of the Tt-Cu A site result in modulation of , which is expected to have an impact on E 0 other than just coulombic stabilization/destabilization. Indeed, it was found that the reduction entropies of the different Tt-Cu A variants, including first and second sphere modifications, increase with .…”

“…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. 53 In all cases, the generated proteins were characterized by a variety of spectroscopic methods such as UV-vis, EPR, NMR, RR and EXAFS, which demonstrate that the structure and the mixed valence character of the Cu A site are preserved in all these variants. Interestingly, a comparison of the UV-vis spectra obtained experimentally and calculated by TD-DFT indicate that the different modifications alter the relative populations of the alternative σ u * and π u ground states, i.e.…”

“…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.…”

Hydrolytic zinc metallopeptides using a computational multi-state design approach

“…17 Although in proteins the extensive network of residue contacts in active sites may compensate for improper modelling of metal-protein interactions, in small scaffolds the chemical environment around the metal ions needs to be properly defined. 18 On this regard, CED approaches are well-suited to address these issues since interactions of the metal ion with both the protein and substrates can be modelled explicitly.…”