Sonodynamic inactivation of gram-negative and gram-positive bacteria in the presence of phenothiazine compounds toluidine blue and azurin A.

BACKGROUND: Sonodynamic antimicrobial chemotherapy (SACT) is an effective antimicrobial treatment that can avoid the production of drug-resistant bacteria. Design and development of new high-efficiency sonosensitizers play a key role in the practical application of SACT. METHODS: The bacteriostatic effects of two phenothiazine compounds, toluidine blue (TB) and azure A (AA) combined with ultrasonic (US) on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were studied, and the sonodynamic antibacterial activities of TB and AA were compared. The reactive oxygen species (ROS) and the types of ROS produced in the sonodynamic system were detected and the sonodynamic mechanisms of TB and AA were proposed. RESULTS: The sonodynamic bacteriostasis mediated by TB and AA increased with the increasing concentration of sonosensitizer, the extension of sonication time and the increase of reaction temperature. The production of ROS was the main reason that TB and AA had excellent sonodynamic antibacterial performance. Singlet oxygen (1O2) and hydroxyl radical (•OH) were the main ROS types in the sonodynamic antibacterial system. The ROS produced by the combined action of AA and US was higher than that of TB. CONCLUSION: Both TB and AA displayed excellent sonodynamic antibacterial activities. Moreover, AA had a higher sonodynamic activity than TB. The electron donation effect and steric hindrance effect of the methyl group of phenothiazine parent nucleus of TB might be the cause of the decrease of its sonodynamic activity. These results would provide a valuable reference for the further study of phenothiazines sonosensitizers and their clinical application in SACT.

Extracellular production of azurin by reusable magnetic Fe3O4 nanoparticle-immobilized Pseudomonas aeruginosa.

Azurin, found in the periplasm of Pseudomonas aeruginosa, has garnered significant attention as a potential anticancer agent in recent years. High-level secretion of proteins into the culture medium, offers a significant advantage over periplasmic or cytoplasmic expression. In this study, for the first time, P. aeruginosa cells were immobilized with magnetic nanoparticles (MNPs) to ensure effective, simple and quick separation of the cells and secretion of periplasmic azurin protein to the culture medium. For this purpose, polyethyleneimine-coated iron oxide (Fe3O4@PEI) MNPs were synthesized and MNPs containing Fe up to 600 ppm were found to be non-toxic to the bacteria. The highest extracellular azurin level was observed in LB medium compared to peptone water. The cells immobilized with 400 ppm Fe-containing MNPs secreted the highest protein. Lastly, the immobilized cells were found suitable for azurin secretion until the sixth use. Thus, the magnetic nanoparticle immobilization method facilitated the release of azurin as well as the simple and rapid separation of cells. This approach, by facilitating protein purification and enabling the reuse of immobilized cells, offers a cost-effective means of protein production, reducing waste cell formation, and thus presents an advantageous method.

Single-step purification and characterization of Pseudomonas aeruginosa azurin.

Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification. Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease, azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of azurin was tested in HeLa cells.

Theoretical insights into the reduction of Azurin metal site with unnatural amino acid substitutions.

Copper-containing proteins play crucial roles in biological systems. Azurin is a copper-containing protein which has a Type 1 copper site that facilitates electron transfer in the cytochrome chain. Previous research has highlighted the significant impact of mutations in the axial Met121 of the copper site on the reduction potential. However, the mechanism of this regulation has not been fully established. In this study, we employed theoretical modeling to investigate the reduction of the Type 1 copper site, focusing on how unnatural amino acid substitutions at Met121 influence its behavior. Our findings demonstrated a strong linear correlation between electrostatic interactions and the reduction potential of the copper site, which indicates that the perturbation of the reduction potential is primarily influenced by electrostatic interactions between the metal ion and the ligating atom. Furthermore, we found that CF/π and CF…H interactions could induce subtle changes in geometry and hence impact the electronic properties of the systems under study. In addition, our calculations suggest the coordination mode and ion-ligand distance could significantly impact the reduction potential of a copper site. Overall, this study offers valuable insights into the structural and electronic properties of the Type 1 copper site, which could potentially guide the design of future artificial catalysts.

Coherent spin transport in a copper protein.

CONTEXT: The coherent electron/spin transport in azurin, a species of copper protein, was calculated based on the Landauer model. The research is motivated by the fast electron transport and spin selectivity/polarization in azurin, which have been reported in relation to the chiral-induced spin selectivity of the peptide structure. The calculated spin polarization of copper proteins was large. This phenomenon was strongly influenced by the spin density of the atoms in the ligand group, whereas the contribution of copper was negligible. The results suggest that spin polarization in copper proteins is enhanced by that of the ligand groups. The predicted spin polarization aligns primarily with the scanning tunneling microscope-based break-junction technique to study the electronic properties of single-molecule junctions. METHODS: Computational techniques employed in this study are nonequilibrium Green's functions (NEGF) and density functional theory (DFT) based on the Landauer model, implemented using the QuantumATK software (Synopsys Inc.). The Perdew-Burke-Ernzerhof (PBE) exchange-correlation functional was adopted for spin-polarized generalized gradient approximation (SGGA). The valence atomic orbitals were constructed using the wavefunctions of the SIESTA package, which was based on the norm-conserving Troullier-Martins relativistic pseudopotentials for describing core electrons. The mesh used for real-space integration was 150 Ha.

Kinetic, electrochemical and spectral characterization of bacterial and archaeal rusticyanins; unexpected stability issues and consequences for applications in biotechnology.

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite.

HYSCORE and QM/MM Studies of Second Sphere Variants of the Type 1 Copper Site in Azurin: Influence of Mutations on the Hyperfine Couplings of Remote Nitrogens.

Secondary coordination sphere (SCS) interactions have been shown to play important roles in tuning reduction potentials and electron transfer (ET) properties of the Type 1 copper proteins, but the precise roles of these interactions are not fully understood. In this work, we examined the influence of F114P, F114N, and N47S mutations in the SCS on the electronic structure of the T1 copper center in azurin (Az) by studying the hyperfine couplings of (i) histidine remote Nε nitrogens and (ii) the amide Np using the two-dimensional (2D) pulsed electron paramagnetic resonance (EPR) technique HYSCORE (hyperfine sublevel correlation) combined with quantum mechanics/molecular mechanics (QM/MM) and DLPNO-CCSD calculations. Our data show that some components of hyperfine tensor and isotropic coupling in N47SAz and F114PAz (but not F114NAz) deviate by up to ∼±20% from WTAz, indicating that these mutations significantly influence the spin density distribution between the CuII site and coordinating ligands. Furthermore, our calculations support the assignment of Np to the backbone amide of residue 47 (both in Asn and Ser variants). Since the spin density distributions play an important role in tuning the covalency of the Cu-Scys bond of Type 1 copper center that has been shown to be crucial in controlling the reduction potentials, this study provides additional insights into the electron spin factor in tuning the reduction potentials and ET properties.

Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features.

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.

Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

Chemical protein synthesis enabled engineering of saccharide oxidative cleavage activity in artificial metalloenzymes.

Chemical protein (semi-)synthesis is a powerful technique allowing the incorporation of unnatural functionalities at any desired protein site. Herein we describe a facile one-pot semi-synthetic strategy for the construction of a type 2 copper center in the active site of azurin, which is achieved by substitution of Met121 with unnatural amino acid residues bearing a strong ligand N,N-bis(pyridylmethyl)amine (DPA) to mimic the function of typical histidine brace-bearing copper monooxygenases, such as lytic polysaccharide monooxygenases (LPMOs) involved in polysaccharide breakdown. The semi-synthetic proteins were routinely obtained in over 10-mg scales to allow for spectroscopic measurements (UV-Vis, CD, and EPR), which provides structural evidences for the CuII-DPA-modified azurins. 4-nitrophenyl-ß-D-glucopyranoside (PNPG) was used as a model substrate for the H2O2-driven oxidative cleavage reaction facilitated by semi-synthetic azurins, and the CuII-6 complex showed a highest activity (TTN 253). Interestingly, our semi-synthetic azurins were able to tolerate high H2O2 concentrations (up to 4000-fold of the enzyme), making them promising for practical applications. Collectively, we establish that chemical protein synthesis can be exploited as a reliable technology in affording large quantities of artificial metalloproteins to facilitate the transformation of challenging chemical reactions.

Tryptophan to Tryptophan Hole Hopping in an Azurin Construct.

Electron transfer (ET) between neutral and cationic tryptophan residues in the azurin construct [ReI(H126)(CO)3(dmp)](W124)(W122)CuI (dmp = 4,7-Me2-1,10-phenanthroline) was investigated by Born-Oppenheimer quantum-mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) simulations. We focused on W124•+ ← W122 ET, which is the middle step of the photochemical hole-hopping process *ReII(CO)3(dmp•-) ← W124 ← W122 ← CuI, where sequential hopping amounts to nearly 10,000-fold acceleration over single-step tunneling (ACS Cent. Sci. 2019, 5, 192-200). In accordance with experiments, UKS-DFT QM/MM/MD simulations identified forward and reverse steps of W124•+ ↔ W122 ET equilibrium, as well as back ET ReI(CO)3(dmp•-) → W124•+ that restores *ReII(CO)3(dmp•-). Strong electronic coupling between the two indoles (≥40 meV in the crossing region) makes the productive W124•+ ← W122 ET adiabatic. Energies of the two redox states are driven to degeneracy by fluctuations of the electrostatic potential at the two indoles, mainly caused by water solvation, with contributions from the protein dynamics in the W122 vicinity. ET probability depends on the orientation of Re(CO)3(dmp) relative to W124 and its rotation diminishes the hopping yield. Comparison with hole hopping in natural systems reveals structural and dynamics factors that are important for designing efficient hole-hopping processes.

Circular permutation at azurin's active site slows down its folding.

Circular permutation (CP) is a technique by which the primary sequence of a protein is rearranged to create new termini. The connectivity of the protein is altered but the overall protein structure generally remains unperturbed. Understanding the effect of CP can help design robust proteins for numerous applications such as in genetic engineering, optoelectronics, and improving catalytic activity. Studies on different protein topologies showed that CP usually affects protein stability as well as unfolding rates. Though a significant number of proteins contain metals or other cofactors, reports of metalloprotein CPs are rare. Thus, we chose a bacterial metalloprotein, azurin, and its CP within the metal-binding site (cpF114). We studied the stabilities, folding, and unfolding rates of apo- and Zn2+-bound CP azurin using fluorescence and circular dichroism. The introduced CP had destabilizing effects on the protein. Also, the folding of the Zn2+-CP protein was much slower than that of the Zn2+-WT or apo-protein. We compared this study to our previously reported azurin-cpN42, where we had observed an equilibrium and kinetic intermediate. cpF114 exhibits an apparent two-state equilibrium unfolding but has an off-pathway kinetic intermediate. Our study hinted at CP as a method to modify the energy landscape of proteins to alter their folding pathways. WT azurin, being a faster folder, may have evolved to optimize the folding rate of metal-bound protein compared to its CPs, albeit all of them have the same structure and function. Our study underscores that protein sequence and protein termini positions are crucial for metalloproteins. TOC Figure. (Top) Zn2+-azurin WT structure (PDB code: 1E67) and 2-D topology diagram of Zn2+-cpF114 azurin. (Bottom) Cartoon diagram representing folding (red arrows) and unfolding (blue arrows) of apo- and Zn2+- WT and cpF114 azurins. The width of the arrows represents the rate of the corresponding processes.

Structural Basis for the Effects of Phenylalanine on Tuning the Reduction Potential of Type 1 Copper in Azurin.

In order to investigate the effects of the secondary coordination sphere in fine-tuning redox potentials (E°') of type 1 blue copper (T1Cu) in cupredoxins, we have introduced M13F, M44F, and G116F mutations both individually and in combination in the secondary coordination sphere of the T1Cu center of azurin (Az) from Pseudomonas aeruginosa. These variants were found to differentially influence the E°' of T1Cu, with M13F Az decreasing E°', M44F Az increasing E°', and G116F Az showing a negligible effect. In addition, combining the M13F and M44F mutations increases E°' by 26 mV relative to WT-Az, which is very close to the combined effect of E°' by each mutation. Furthermore, combining G116F with either M13F or M44F mutation resulted in negative and positive cooperative effects, respectively. Crystal structures of M13F/M44F-Az, M13F/G116F-Az, and M44F/G116F-Az combined with that of G116F-Az reveal these changes arise from steric effects and fine-tuning of hydrogen bond networks around the copper-binding His117 residue. The insights gained from this study would provide another step toward the development of redox-active proteins with tunable redox properties for many biological and biotechnological applications.

Systematic elucidation of the second coordination sphere effect on the structure and properties of a blue copper protein, pseudoazurin.

The rational structural and computational studies of a blue copper protein, pseudoazurin (PAz), and its Met16X (X = Phe, Leu, Val, Ile) variants gave clear functional meanings of the noncovalent interaction (NCI) through the second coordination sphere. The high-resolution X-ray crystal structures of Met16X PAz demonstrated that the active site geometry is significantly affected by the substitution of Met16, which is located within the NCI distance from the His81 imidazole ring at the copper active site. The computational chemistry calculations based on the crystal structure analyses confirmed that the NCI of S-π/CH-π (wild-type), π-π (Met16Phe), double CH-π (Met16Leu), and single CH-π (Met16Val and Met16Ile). The estimated interaction energies for the NCI demonstrated that the fine-tuning of the protein stability and Cu site properties form the second coordination sphere of PAz.

Role of Metal Cofactor in Enhanced Thermal Stability of Azurin.

Metal cofactors are critical centers for different biochemical processes of metalloproteins, and often, this metal coordination renders additional structural stability. In this study, we explore the additional stability conferred by the copper ion on azurin by analyzing both the apo and holo forms using temperature replica exchange molecular dynamics (REMD) data. We find a 14 K decrease in denaturation temperature for apo (406 K) azurin relative to that of holo (420 K), indicating a copper ion-induced additional thermal stability for holo azurin. The unfolding of apo azurin begins with the melting of α-helix and ß-sheet V, similar to that of holo form. ß-Sheets IV, VII, and VIII are comparatively more stable than other ß-strands and melt at higher temperatures. Similar to holo azurin, the strong hydrophobic interactions among the apolar residues in the protein core is the key factor that renders high stability to apo protein as well. We construct free energy surfaces at different temperatures to capture the major conformations along the unfolding basins of the protein. Using contact maps from different basins we show the changes in the interaction between different residues along the unfolding pathway. Furthermore, we compare the Cα root-mean-square fluctuations (Cα-RMSF) and B-factor of all residues of apo and holo forms to understand the flexibility of different regions. The concerted displacement of α-helix and ß-sheets V and VI from the protein core is another distinction we observe for apo compared to the holo form, where ß-sheet VI was relatively stable.

Applicability of perturbed matrix method for charge transfer studies at bio/metallic interfaces: a case of azurin.

As the field of nanoelectronics based on biomolecules such as peptides and proteins rapidly grows, there is a need for robust computational methods able to reliably predict charge transfer properties at bio/metallic interfaces. Traditionally, hybrid quantum-mechanical/molecular-mechanical techniques are employed for systems where the electron hopping transfer mechanism is applicable to determine physical parameters controlling the thermodynamics and kinetics of charge transfer processes. However, these approaches are limited by a relatively high computational cost when extensive sampling of a configurational space is required, like in the case of soft biomatter. For these applications, semi-empirical approaches such as the perturbed matrix method (PMM) have been developed and successfully used to study charge-transfer processes in biomolecules. Here, we explore the performance of PMM on prototypical redox-active protein azurin in various environments, from solution to vacuum interfaces with gold surfaces and protein junction. We systematically benchmarked the robustness and convergence of the method with respect to the quantum-centre size, size of the Hamiltonian, number of samples, and level of theory. We show that PMM can adequately capture all the trends associated with the structural and electronic changes related to azurin oxidation at bio/metallic interfaces.

Structural basis of bacterial effector protein azurin targeting tumor suppressor p53 and inhibiting its ubiquitination.

Tumor suppressor p53 prevents tumorigenesis by promoting cell cycle arrest and apoptosis through transcriptional regulation. Dysfunction of p53 occurs frequently in human cancers. Thus, p53 becomes one of the most promising targets for anticancer treatment. A bacterial effector protein azurin triggers tumor suppression by stabilizing p53 and elevating its basal level. However, the structural and mechanistic basis of azurin-mediated tumor suppression remains elusive. Here we report the atomic details of azurin-mediated p53 stabilization by combining X-ray crystallography with nuclear magnetic resonance. Structural and mutagenic analysis reveals that the p28 region of azurin, which corresponds to a therapeutic peptide, significantly contributes to p53 binding. This binding stabilizes p53 by disrupting COP1-mediated p53 ubiquitination and degradation. Using the structure-based design, we obtain several affinity-enhancing mutants that enable amplifying the effect of azurin-induced apoptosis. Our findings highlight how the structure of the azurin-p53 complex can be leveraged to design azurin derivatives for cancer therapy.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key ß-barrel, ß-sandwich, ß-α-ß, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48.

Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (E°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and E° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E° and reveal properties of the protein microenvironment surrounding tryptophan.

Theory of Protein Charge Transfer: Electron Transfer between Tryptophan Residue and Active Site of Azurin.

One reaction step in the conductivity relay of azurin, electron transfer between the Cu-based active site and the tryptophan residue, is studied theoretically and by classical molecular dynamics simulations. Oxidation of tryptophan results in electrowetting of this residue. This structural change makes the free energy surfaces of electron transfer nonparabolic as described by the Q-model of electron transfer. We analyze the medium dynamical effect on protein electron transfer produced by coupled Stokes-shift dynamics and the dynamics of the donor-acceptor distance modulating electron tunneling. The equilibrium donor-acceptor distance falls in the plateau region of the rate constant, where it is determined by the protein-water dynamics, and the probability of electron tunneling does not affect the rate. The crossover distance found here puts most intraprotein electron-transfer reactions under the umbrella of dynamical control. The crossover between the medium-controlled and tunneling-controlled kinetics is combined with the effect of the protein-water medium on the activation barrier to formulate principles of tunability of protein-based charge-transfer chains. The main principle in optimizing the activation barrier is the departure from the Gaussian-Gibbsian statistics of fluctuations promoting activated transitions. This is achieved either by incomplete (nonergodic) sampling, breaking the link between the Stokes-shift and variance reorganization energies, or through wetting-induced structural changes of the enzyme's active site.