Outer-Sphere Contributions to the Electronic Structure of Type Zero Copper Proteins

Abstract

Bioinorganic canon states that active-site thiolate coordination promotes rapid electron transfer (ET) to and from type 1 copper proteins. In recent work, we have found that copper ET sites in proteins also can be constructed without thiolate ligation (called “type zero” sites). Here we report multifrequency electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and nuclear magnetic resonance (NMR) spectroscopic data together with density functional theory (DFT) and spectroscopy-oriented configuration interaction (SORCI) calculations for type zero Pseudomonas aeruginosa azurin variants. Wild-type (type 1) and type zero copper centers experience virtually identical ligand fields. Moreover, O-donor covalency is enhanced in type zero centers relative that in the C112D (type 2) protein. At the same time, N-donor covalency is reduced in a similar fashion to type 1 centers. QM/MM and SORCI calculations show that the electronic structures of type zero and type 2 are intimately linked to the orientation and coordination mode of the carboxylate ligand, which in turn is influenced by outer-sphere hydrogen bonding.

Spin Delocalization Over Type Zero Copper

“…This compares quite well with the Löwdin spin populations obtained from density functional theory (DFT) calculations of C112D, 0.071 for H46 and 0.055 for H117. 52 The histidines’ A( 14 N) values of the type zero Cu II in C112D/M121X(X=L,F) are closer to those of WT azurin and using the above arguments we obtain for X=L ρ N1 ~0.06 and ρ N2 ~0.04 and for X=F ρ N1 ~ ρ N2 ~0.04. The predicted DFT values 52 are ρ N =0.05, 0.013 for X=L and ρ N =0.047, 0.040 for X=F, for H46 and H117, respectively.…”

“…52 The histidines’ A( 14 N) values of the type zero Cu II in C112D/M121X(X=L,F) are closer to those of WT azurin and using the above arguments we obtain for X=L ρ N1 ~0.06 and ρ N2 ~0.04 and for X=F ρ N1 ~ ρ N2 ~0.04. The predicted DFT values 52 are ρ N =0.05, 0.013 for X=L and ρ N =0.047, 0.040 for X=F, for H46 and H117, respectively. Again the values compare well with DFT ones, except for H117 of C112D/M121L.…”

“…DFT calculation gave for the optimized structures of C112D/M121L and C112D a finite isotropic hyperfine constants around 1.5 MHz and 0.6–0.9 MHz for the exchangeable ring proton H ε2 of H46 and H117, respectively. 52 Thus, this coupling could arise from H46 ring. The proton couplings observed do not deviate significantly from those expected from dipolar interactions and do not reveal any large unexpected spin density that would be manifested by a large isotropic hyperfine coupling as observed for the cysteine β-protons in type 1 copper.…”

Biological Outer-Sphere Coordination

“…Moreover, the Cu-O e -C g -C b dihedral decreases from 40 to 3 . This monodentate coordination has been proposed to give rise to the type zero electronic structure and is the subject of an upcoming report [53]. Fig.…”

Modular Artificial Cupredoxins

“…33,34 New methods we introduce here use the spectra from two microwave frequencies, paired with simulation, to determine the A-tensors in the absence of resolved hyperfine splittings. The EPR spectra for 2a showed a 4-line hyperfine pattern at g z = 2.19 with a reduced A z value of 353 MHz compared to the average A z value of 523 MHz obtained for 1a .…”