Remote Water-Mediated Proton Transfer Triggers Inter-Cu Electron Transfer: Nitrite Reduction Activation in Copper-Containing Nitrite Reductase.

The copper-containing nitrite reductase (CuNiR) catalyzes the biological conversion of nitrite to nitric oxide; key long-range electron/proton transfers are involved in the catalysis. However, the details of the electron-/proton-transfer mechanism are still unknown. In particular, the driving force of the electron transfer from the type-1 copper (T1Cu) site to the type-2 copper (T2Cu) site is ambiguous. Here, we explored the two possible proton-transfer channels, the high-pH proton channel and the primary proton channel, by using two-layered ONIOM calculations. Our calculation results reveal that the driving force for electron transfer from T1Cu to T2Cu comes from a remote water-mediated triple-proton-coupled electron-transfer mechanism. In the high-pH proton channel, the water-mediated triple-proton transfer occurs from Glu113 to an intermediate water molecule, whereas in the primary channel, the transfer is from Lys128 to His260. Subsequently, the two channels employ another two or three distinct proton-transfer steps to deliver the proton to the nitrite substrate at the T2Cu site. These findings explain the detailed proton-/electron-transfer mechanisms of copper-containing nitrite reductase and could extend our understanding of the diverse proton-coupled electron-transfer mechanisms in complicated proteins.

Designed azurins show lower reorganization free energies for intraprotein electron transfer.

Low reorganization free energies are necessary for fast electron transfer (ET) reactions. Hence, rational design of redox proteins with lower reorganization free energies has been a long-standing challenge, promising to yield a deeper understanding of the underlying principles of ET reactivity and to enable potential applications in different energy conversion systems. Herein we report studies of the intramolecular ET from pulse radiolytically produced disulfide radicals to Cu(II) in rationally designed azurin mutants. In these mutants, the copper coordination sphere has been fine-tuned to span a wide range of reduction potentials while leaving the metal binding site effectively undisrupted. We find that the reorganization free energies of ET within the mutants are indeed lower than that of WT azurin, increasing the intramolecular ET rate constants almost 10-fold: changes that are correlated with increased flexibility of their copper sites. Moreover, the lower reorganization free energy results in the ET rate constants reaching a maximum value at higher driving forces, as predicted by the Marcus theory.

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.

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.

Resonance Raman spectra of blue copper proteins: Variable temperature spectra of Thermus thermophilus HB27 laccase.

The resonance Raman (rR) spectra of the oxidized type 1 copper active site (CuT1) in Thermus thermophilus HB27 laccase (Tth-lac) has been determined in the 20 to 80 °C temperature range using 633-nm excitation. The positions and relative intensities of rR peaks are virtually independent of temperature, indicating that CuT1 ligation is robust over the investigated range. The intensity-weighted average of Tth-lac Cu-SCys vibrations (<ν(Cu-SCys)>) = 423 cm-1) is higher than those of most cupredoxins but is comparable to those of other multicopper oxidases (MCOs). <ν(Cu-SCys)> values for Tth-lac and several CuT1 centers in cupredoxins and MCOs do not correlate well with Cu-SCys bond lengths but do exhibit systematic trends with redox thermodynamic properties. PROLOGUE: F. Ann Walker was a great scholar and dear friend. While at Columbia in the early 1960s, I (HBG) followed her graduate work at Brown on the effects of axial ligands on vanadyl ion EPR spectra. Dick Carlin, her thesis adviser, invited me to serve as external member of her thesis committee. I joined, made my way to Providence, met her just before the exam, and greatly admired (enjoyed!) her thoughtful responses to questions from physical chemists about metal-oxo electronic structures. Our friendship grew stronger over the years, enhanced by lively discussions of heme protein chemistry in San Francisco, Pasadena, Tucson, and at Gordon Research Conferences. Ann was a superstar in biological inorganic chemistry. She will be sorely missed but not forgotten.

Putative role of conserved water molecules in the hydration and inter-domain recognition of mono nuclear copper centers in O2-bound human ceruloplasmin: A comparative study between X-ray and MD simulated structures.

Human Ceruloplasmin (hCP) is an unique multicopper oxidase which involves in different biological functions e.g., iron metabolism, copper transportation, biogenic amine oxidation ,and its malfunction causes Wilson's and Menkes diseases. MD- simulation studies of O2- bound solvated structure have revealed the role of several conserved/ semi-conserved water molecules in the hydration of type-I copper centers and their involvement to recognition dynamics of these metal centers. In O2- bound structure, hydration potentiality of CuRS (Cu1106) type-I copper center is observed to be unique, where two water molecules (W1-W2) are interacting with the metal sites, which was not found in X-ray structures of hCP. Generally, in the interdomain recognition of CuCys-His to CuRS, CuRS to CuPR and CuPR to CuCys-His centers, the copper bound His-residue of one domain interacts with the Glu-residue of other complementary domain through conserved/ semi-conserved (W3 to W5) water- mediated hydrogen bonds (Cu-His...W...Glu), however direct salt-bridge (Cu-His...Glu) interaction were observed in the X- ray structures. The MD- simulated and X- ray structures have indicated some possibilities on the Cu-His...W...Glu ↔ Cu-His...Glu transition during the interdomain recognition of type-I copper centers, which may have some importance in biology and chemistry of ceruloplasmin.

The type 1 copper site of pseudoazurin: axial and rhombic.

We report on a high-frequency electron-paramagnetic-resonance study of the type 1 copper site of pseudoazurin. The spectra fully resolve the contribution of a nearly axial spectrum besides the rhombic spectrum, which unequivocally proves the existence of two conformations of the copper site. Pseudoazurins have been considered from Achromobacter cycloclastes including eight mutants and from Alcaligenes faecalis. The two conformations are virtually the same for all pseudoazurins, but the rhombic/axial population varies largely, between 91/9 and 33/67. These observations are discussed in relation to optical absorption spectra and X-ray diffraction structures. A similar observation for fern plastocyanin from Dryopteris crassirhizoma suggests that dual conformations of type 1 copper sites are more common.

Structural and redox properties of the small laccase Ssl1 from Streptomyces sviceus.

UNLABELLED: Laccases (EC 1.10.3.2) are members of the multicopper oxidase family. They oxidize diverse electron-rich substrates through electron abstraction by the type 1 copper ion in the enzyme active site. Abstracted electrons are transferred to the trinuclear copper cluster, where molecular oxygen serves as final acceptor and is reduced to water. Laccase activity is assumed to depend on the redox potential of its type 1 copper ion. Whereas numerous studies have been undertaken to elucidate the determinants of the redox potential of type 1 copper ions in one-domain cupredoxins and in three-domain laccases, such experimental investigations are lacking for recently described, small, two-domain laccases. In this work, the crystal structure of the small laccase Ssl1 from Streptomyces sviceus was solved, and the positions that might influence the redox potential of Ssl1 were depicted. On the basis of this knowledge, several Ssl1 variants were constructed with an increase in redox potential of 16-81 mV, from 375 mV to 391-456 mV. Mutation of residues in close proximity to the type 1 copper center resulted in a predicted increase in the redox potential of the copper center; however, there was a reduced specific activity for the oxidation of 2,6-dimethoxyphenol, which has a relatively low redox potential. Mutations more distant to the type 1 copper also led to an increased redox potential of the copper center, and resulted in variants able to oxidize the high redox potential substrates 1,2-dihydroxyanthraquinone-3-sulfonic acid (Alizarin Red S) and indigo carmine more efficiently than wild-type Ssl1. DATABASE: The atomic coordinates of the structure of Ssl1 laccase from Streptomyces sviceus and structure factors have been deposited in the RCSB Protein Data Bank (4M3H) STRUCTURED DIGITAL ABSTRACT: •Ssl1 and Ssl1 bind by x-ray crystallography (View interaction).

Direct electrochemistry and intramolecular electron transfer of ascorbate oxidase confined on L-cysteine self-assembled gold electrode.

A direct electrochemistry and intramolecular electron transfer of multicopper oxidases are of a great importance for the fabrication of these enzyme-based bioelectrochemical-devices. Ascorbate oxidase from Acremonium sp. (ASOM) has been successfully immobilized via a chemisorptive interaction on the l-cysteine self-assembled monolayer modified gold electrode (cys-SAM/AuE). Thermodynamics and kinetics of adsorption of ASOM on the cys-SAM/AuE were studied using cyclic voltammetry. A well-defined redox wave centered at 166±3mV (vs. Ag│AgCl│KCl(sat.)) was observed in 5.0mM phosphate buffer solution (pH7.0) at the fabricated ASOM electrode, abbreviated as ASOM/cys-SAM/AuE, confirming a direct electrochemistry, i.e., a direct electron transfer (DET) between ASOM and cys-SAM/AuE. The direct electrochemistry of ASOM was further confirmed by taking into account the chemical oxidation of ascorbic acid (AA) by O2 via an intramolecular electron transfer in the ASOM as well as the electrocatalytic oxidation of AA at the ASOM/cys-SAM/AuE. Thermodynamics and kinetics of the adsorption of ASOM on the cys-SAM/AuE have been elaborated along with its direct electron transfer at the modified electrodes on the basis of its intramolecular electron transfer and electrocatalytic activity towards ascorbic acid oxidation and O2 reduction. ASOM saturated surface area was obtained as 2.41×10(-11)molcm(-2) with the apparent adsorption coefficient of 1.63×10(6)Lmol(-1). The ASOM confined on the cys-SAM/AuE possesses its essential enzymatic function.

Enhancement of catalysis and functional expression of a bacterial laccase by single amino acid replacement.

Structure-function relationships underlying laccases properties are very limited that makes these enzymes interesting for protein engineering approaches. Therefore in the current study, a thermostable laccase that was isolated from Bacillus sp. HR03 with the ability of bilirubin oxidation besides its laccase and tyrosinase activity is used. The extensive application of this enzyme is limited by its low expression level in Escherichia coli. Based on sequence alignments and structural studies, three single amino acid substitutions, D500G, D500E, D500S and a glycine insertion, are introduced using site-directed mutagenesis to evaluate the role of Asp(500) located in the C-terminal segment close to the T1 copper center. Substitution of aspartic acid with less sterically hindered, conserved residue such as glycine increase kcat (2.3 fold) and total activity (7.3 fold) which is accompanied by a significant increase in the expression level up to 3 fold. Biochemical characterization and structural studies using far-UV CD and fluorescence spectroscopy reveal the importance of C-terminal copper-binding loop in the laccase functional expression and catalytic efficiency. Kinetic characterization of the purified mutants toward 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ) and bilirubin, shows that substrate specificity is left unchanged.