title: Spectroscopic and DFT studies of second-sphere variants of the type 1 copper site in azurin: covalent and nonlocal electrostatic contributions to reduction potentials.
abstract: The reduction potentials (E(0)) of type 1 (T1) or blue copper (BC) sites in proteins and enzymes with identical first coordination spheres around the redox active copper ion can vary by ~400 mV. Here, we use a combination of low-temperature electronic absorption and magnetic circular dichroism, electron paramagnetic resonance, resonance Raman, and S K-edge X-ray absorption spectroscopies to investigate a series of second-sphere variants--F114P, N47S, and F114N in Pseudomonas aeruginosa azurin--which modulate hydrogen bonding to and protein-derived dipoles nearby the Cu-S(Cys) bond. Density functional theory calculations correlated to the experimental data allow for the fractionation of the contributions to tuning E(0) into covalent and nonlocal electrostatic components. These are found to be significant, comparable in magnitude, and additive for active H-bonds, while passive H-bonds are mostly nonlocal electrostatic in nature. For dipoles, these terms can be additive to or oppose one another. This study provides a methodology for uncoupling covalency from nonlocal electrostatics, which, when coupled to X-ray crystallographic data, distinguishes specific local interactions from more long-range protein/active interactions, while affording further insight into the second-sphere mechanisms available to the protein to tune the E(0) of electron-transfer sites in biology.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Ryan G
authors.initials: RG
authors.lastname: Hadt
authors.forename: Ning
authors.initials: N
authors.lastname: Sun
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Marshall
authors.forename: Keith O
authors.initials: KO
authors.lastname: Hodgson
authors.forename: Britt
authors.initials: B
authors.lastname: Hedman
authors.forename: Yi
authors.initials: Y
authors.lastname: Lu
authors.forename: Edward I
authors.initials: EI
authors.lastname: Solomon
mesh_terms: D001400:Azurin / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry*; D018360:Crystallography, X-Ray; D004578:Electron Spin Resonance Spectroscopy; D006860:Hydrogen Bonding; D008958:Models, Molecular; D010084:Oxidation-Reduction; D017354:Point Mutation; D011550:Pseudomonas aeruginosa / Q000737:chemistry* / Q000235:genetics; D013059:Spectrum Analysis, Raman; D055672:Static Electricity; D056928:X-Ray Absorption Spectroscopy
chemical_list: D001400:Azurin; D003300:Copper