Spectroscopic and density functional theory studies of the blue-copper site in M121SeM and C112SeC azurin: Cu-Se versus Cu-S bonding

Abstract

S K-edge X-ray absorption, UV-vis absorption, magnetic circular dichroism (MCD), and resonance Raman spectroscopies are used to investigate the electronic structure differences among WT, M121SeM, and C112SeC Pseudomonas aeruginosa (P.a) azurin. A comparison of S K-edge XAS of WT and M121SeM azurin and a CuII-thioether model complex shows that the 38% S character in the ground state wave function of the blue-copper (BC) sites solely reflects the Cu-SCys bond. Resonance Raman (rR) data on WT and C112SeC azurin give direct evidence for the kinematic coupling between the Cu-SCys stretch and the cysteine deformation modes in WT azurin, which leads to multiple features in the rR spectrum of the BC site. The UV-vis absorption and MCD data on WT, M121SeM, and C112SeC give very similar C0/D0 ratios, indicating that the C-term MCD intensity mechanism involves Cu-centered spin-orbit coupling (SOC). The spectroscopic data combined with density functional theory (DFT) calculations indicate that SCys and SeCys have similar covalent interactions with Cu at their respective bond lengths of 2.1 and 2.3 A. This reflects the similar electronegativites of S and Se in the thiolate/selenolate ligand fragment and explains the strong spectroscopic similarities between WT and C112SeC azurin.

Figure 1

S K-edge X-ray absorption spectra of WT azurin (black line), M121SeM (red line), and C112SeC (green line) azurins. Inset shows the expanded pre-edge region. The spectra have been renormalized in each case to account for noncoordinating S-containing amino acids in the proteins.

Figure 2

S K-edge X-ray absorption spectra of WT azurin (black line) and the Cu–S_thioether complex (CuL1) (blue line). Inset shows the second-derivative spectra. The pre-edge energy positions have been marked in both cases.

Figure 3

Electronic absorption (left panel) and magnetic circular dichroism (right panel) spectra of (A) WT (black line), (B) M121SeM (red line), and (C) C112SeC (green line) azurins. Simultaneous Gaussian fits require eight bands to fit the data, which have been depicted with dashed lines. The bands have been labeled 1–8 for all three proteins.

Figure 4

Resonance Raman spectra obtained upon excitation at 647.1 nm for WT (black line), M121SeM (red line), and C112SeC (green line) azurins. Lines mark the position of the intensity-weighted average of the Cu–S vibrations.

Figure 5

ψ*_β–LUMOof the 141-atom azurin model (the isocontour value is 0.03 a.u.). The contour plots indicate very similar ground-state wave-functions of the three proteins with only small quantitative differences.

Figure 6

TD–DFT-calculated spectra of WT azurin (black dotted line), the C112SeC mutant (red dotted line), [Cu^II(tpz)(SC₆F₅)] (black solid line), and [Cu^II(tpz)(SeC₆F₅)] (red solid line).

Figure 7

DFT calculated Cu–X distortion (arbitrary units) along normal modes for [(tpz)Cu(XC₆F₅)]; X = S (black line), X = Se (blue line), and X = ³²Se (red line).

Figure 8

Interaction diagram for β-spin molecular orbitals of [Cu^II(tpz)-(SeC₆F₅)] with [CuII(tpz)]⁺ and SeC₆F₅⁻ as fragments and the corresponding σ and π interactions are shown in red and blue. The molecular orbitals of the Cu(tpz)⁺ and SeC₆F₅⁻ fragments are shifted by 4.0 and −4.5 eV, respectively. The interaction diagram for β-spin molecular orbitals of [Cu^II-(tpz)(SC₆F₅)] is presented in Figure S4.80 The interaction diagram for α-spin molecular orbitals is very similar. However, since the α-spin Cu d_x²−y² fragment orbital is occupied, there is no net contribution to bonding from the π ligand-to-metal donation from this spin-orbital.