Role of protein frame and solvent for the redox properties of azurin from Pseudomonas aeruginosa

Abstract

We have coupled hybrid quantum mechanics (density functional theory; Car-Parrinello)/molecular mechanics molecular dynamics simulations to a grand-canonical scheme, to calculate the in situ redox potential of the Cu 2؉ ؉ e ؊ 3 Cu ؉ half reaction in azurin from Pseudomonas aeruginosa. An accurate description at atomistic level of the environment surrounding the metal-binding site and finite-temperature fluctuations of the protein structure are both essential for a correct quantitative description of the electronic properties of this system. We report a redox potential shift with respect to copper in water of 0.2 eV (experimental 0.16 eV) and a reorganization free energy ‫؍‬ 0.76 eV (experimental 0.6 -0.8 eV). The electrostatic field of the protein plays a crucial role in fine tuning the redox potential and determining the structure of the solvent. The inner-sphere contribution to the reorganization energy is negligible. The overall small value is mainly due to solvent rearrangement at the protein surface.density functional theory ͉ electron transfer ͉ molecular dynamics ͉ reorganization energy E lectron transfer (ET) processes are ubiquitous chemical reactions that occur in a variety of essential biological functions, such as immune response, respiration, and photosynthesis (1-10). Among the different families of ET proteins, single-copper cupredoxins, also known as blue copper proteins, are particularly appealing for theoretical studies because of their relatively small size and the great availability of experimental measurements in the literature. Blue copper proteins exchange electrons among themselves or with other redox proteins, such as cytochrome c551 or nitrite reductase, using a protein-bound Cu metal ion, that can exist in the Cu(II) or Cu(I) redox states (11,12). The copper ion forms a type 1 Cu-binding site ( Fig. 1), which is characterized by a bright blue color, a narrow hyperfine splitting in the electron paramagnetic resonance (EPR) spectra, a high reduction potential (13-15), and a strong structural similarity between oxidized and reduced states (13-17). In fact, in both oxidation states, the copper ion is coordinated by a cysteine (Cys) thiolate group and two histidine (His) nitrogen atoms in a trigonal planar conformation. The coordination polyhedron is completed by one axial ligand, typically a methionine (Met) thioether group. In azurin, a backbone amide oxygen of a glycine (Gly) constitutes an additional axial ligand (18). The structural similarity between the two redox states provides an advantage for the functionality of these ET proteins, as the reorganization free energy () for the redox process is small [0.6-0.8 eV (17, 19)], allowing a high ET rate (20-23). This is not the case for solvated copper ions and synthetic Cu complexes, which tend to have large structural changes concurrent with any variation in their oxidation state (24). The origin of the low value of is still unclear. The initial hypothesis implied that the rigidity of the protein would force Cu(II) to be bound in a geomet...

Fundamental signatures of short- and long-range electron transfer for the blue copper protein azurin at Au/SAM junctions

“…This agreement suggests that the value of the reorganization free energy mostly comes from water that is distant from the active site hydrophobic patch. Indeed, the rather small value of λ o ≈ 0.3 eV found in this work and by others (19,20,26,31) is considerably smaller than that one finds from various homogeneous experiments (63,64) or theoretical calculations (11). This difference may result from partial burial of the protein's active site into the SAM's outer layer, hence its essential isolation from nearby water.…”

“…This difference may result from partial burial of the protein's active site into the SAM's outer layer, hence its essential isolation from nearby water. Adjacent water is believed to contribute strongly to λ o , especially to its entropic part (11), and one might expect such contributions to be negligible for hydrophobically adsorbed Az, and lead to ΔS Ã a ≈ 0. Importantly, a similar conclusion is deducible for ET within the comparable Au/SAM systems with covalently attached nonbiological metal complexes acting as redox probes (46,50).…”

“…T he intrinsic electron transfer (ET) mechanisms of even small and otherwise well-characterized proteins such as cytochrome c or azurin (Az) are difficult to identify conclusively because of the proteins' complexity, i.e., inhomogeneous structural and dynamic properties (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The use of bioelectrochemical tunneling junctions, such as self-assembled monolayer (SAM) films of variable composition and thickness on metal electrodes, with redox proteins immobilized at the solution interface (or freely diffusing to the SAM terminal groups) have been shown to provide an assembly with well-defined and variable control parameters.…”

Protein electron transfer: Dynamics and statistics

“…65 It is typically small for cofactors participating in protein electron transfer. 69 It has been argued that the combination of a rigid structure of the cofactor with delocalization of the transferred electron over a number of its atoms helps to reduce intramolecular reorganization 70 and, by that, the energy penalty of elastic deformation caused by transferring the electron.…”

Catalytic Cycle of Multicopper Oxidases Studied by Combined Quantum- and Molecular-Mechanical Free-Energy Perturbation Methods

“…However, to obtain information on the influence of the surrounding protein, more sophisticated methods are needed. 30,32,[34][35][36][37][38] In particular, several groups have estimated and rationalised the redox potentials of Cu T1 sites in proteins 36,[39][40][41][42] and the TNC in MCOs has also been studied. 43 Likewise, many methods have been developed to calculate acidity constants, both of small molecules in solution and of various groups in proteins.…”