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Inorganic Chemistry Jul 2019Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in...
Nickel-containing enzymes such as methyl coenzyme M reductase (MCR) and carbon monoxide dehydrogenase/acetyl coenzyme A synthase (CODH/ACS) play a critical role in global energy conversion reactions, with significant contributions to carbon-centered processes. These enzymes are implied to cycle through a series of nickel-based organometallic intermediates during catalysis, though identification of these intermediates remains challenging. In this work, we have developed and characterized a nickel-containing metalloprotein that models the methyl-bound organometallic intermediates proposed in the native enzymes. Using a nickel(I)-substituted azurin mutant, we demonstrate that alkyl binding occurs via nucleophilic addition of methyl iodide as a methyl donor. The paramagnetic Ni-CH species initially generated can be rapidly reduced to a high-spin Ni-CH species in the presence of exogenous reducing agent, following a reaction sequence analogous to that proposed for ACS. These two distinct bioorganometallic species have been characterized by optical, EPR, XAS, and MCD spectroscopy, and the overall mechanism describing methyl reactivity with nickel azurin has been quantitatively modeled using global kinetic simulations. A comparison between the nickel azurin protein system and existing ACS model compounds is presented. Ni-CH Az is only the second example of two-electron addition of methyl iodide to a Ni center to give an isolable species and the first to be formed in a biologically relevant system. These results highlight the divergent reactivity of nickel across the two intermediates, with implications for likely reaction mechanisms and catalytically relevant states in the native ACS enzyme.
Topics: Acetyl Coenzyme A; Azurin; Catalysis; Chromatography, Gas; Gene Expression Regulation, Bacterial; Kinetics; Magnetic Phenomena; Mutation; Nickel; Organometallic Compounds; Pseudomonas aeruginosa; Spectrum Analysis
PubMed: 30788970
DOI: 10.1021/acs.inorgchem.8b03546 -
Journal of Materials Science. Materials... Sep 2019Metal-on-metal (MoM) hip arthroplasties produce abundant implant-derived wear debris composed mainly of cobalt (Co) and chromium (Cr). Cobalt-chromium (Co-Cr) wear...
Metal-on-metal (MoM) hip arthroplasties produce abundant implant-derived wear debris composed mainly of cobalt (Co) and chromium (Cr). Cobalt-chromium (Co-Cr) wear particles are difficult to identify histologically and need to be distinguished from other wear particle types and endogenous components (e.g., haemosiderin, fibrin) which may be present in MoM periprosthetic tissues. In this study we sought to determine whether histological stains that have an affinity for metals are useful in identifying Co-Cr wear debris in MoM periprosthetic tissues. Histological sections of periprosthetic tissue from 30 failed MoM hip arthroplasties were stained with haematoxylin-eosin (HE), Solochrome Cyanine (SC), Solochrome Azurine (SA) and Perls' Prussian Blue (PB). Sections of periprosthetic tissue from 10 cases of non-MoM arthroplasties using other implant biomaterials, including titanium, ceramic, polymethylmethacrylate (PMMA) and ultra-high molecular weight polyethylene (UHMWP) were similarly analysed. Sections of 10 cases of haemosiderin-containing knee tenosynovial giant cell tumour (TSGCT) were also stained with HE, SC, SA and PB. In MoM periprosthetic tissues, SC stained metal debris in phagocytic macrophages and in the superficial necrotic zone which exhibited little or no trichrome staining for fibrin. In non-MoM periprosthetic tissues, UHMWP, PMMA, ceramic and titanium particles were not stained by SC. Prussian Blue, but not SC or SA, stained haemosiderin deposits in MoM periprosthetic tissues and TSGT. Our findings show that SC staining (most likely Cr-associated) is useful in distinguishing Co-Cr wear particles from other metal/non-metal wear particles types in histological preparations of periprosthetic tissue and that SC reliably distinguishes haemosiderin from Co-Cr wear debris.
Topics: Arthroplasty, Replacement, Hip; Azurin; Benzenesulfonates; Chromium; Coloring Agents; Eosine Yellowish-(YS); Equipment Failure Analysis; Ferrocyanides; Giant Cells, Foreign-Body; Hematoxylin; Hip Joint; Hip Prosthesis; Histological Techniques; Humans; Macrophages; Metal Nanoparticles; Metal-on-Metal Joint Prostheses; Polyethylenes; Staining and Labeling
PubMed: 31493091
DOI: 10.1007/s10856-019-6304-0 -
Chemical Science Nov 2019Monitoring the fluorescence of single-dye-labeled azurin molecules, we observed the reaction of azurin with hexacyanoferrate under controlled redox potential yielding...
Monitoring the fluorescence of single-dye-labeled azurin molecules, we observed the reaction of azurin with hexacyanoferrate under controlled redox potential yielding data on the timing of individual (forward and backward) electron transfer (ET) events. Change-point analysis of the time traces demonstrates significant fluctuations of ET rates and of mid-point potential . These fluctuations are a signature of dynamical heterogeneity, here observed on a 14 kDa protein, the smallest to date. By correlating changes in forward and backward reaction rates we found that 6% of the observed change events could be explained by a change in midpoint potential, while for 25% a change of the donor-acceptor coupling could explain the data. The remaining 69% are driven by variations in complex association constants or structural changes that cause forward and back ET rates to vary independently. Thus, the observed spread in individual ET rates could be related in a unique way to variations in molecular parameters. The relevance for the understanding of metabolic processes is briefly discussed.
PubMed: 34123050
DOI: 10.1039/c9sc05405g -
Journal of Inorganic Biochemistry Sep 2022Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino...
Amicyanin is a type 1 copper protein with a single tryptophan residue. Using genetic code expansion, the tryptophan was selectively replaced with the unnatural amino acid, 5-hydroxytryptophan (5-HTP). The 5-HTP substituted amicyanin exhibited absorbance at 300-320 nm, characteristic of 5-HTP and not seen in native amicyanin. The fluorescence emission maximum in 5-HTP substituted amicyanin is redshifted from 318 nm in native amicyanin to 331 nm and to 348 nm in the unfolded protein. The fluorescence quantum yield of 5-HTP substituted amicyanin mutant was much less than that of native amicyanin. Differences in intrinsic fluorescence are explained by differences in the excited states of tryptophan versus 5-HTP and the intraprotein environment. The substitution of tryptophan with 5-HTP did not affect the visible absorbance and redox potential of the copper, which is 10 Å away. In amicyanin and other cupredoxins, an unexplained quenching of the intrinsic fluorescence by the bound copper is observed. However, the fluorescence of 5-HTP substituted amicyanin is not quenched by the copper. It is shown that the mechanism of quenching in native amicyanin is Förster, or fluorescence, resonance energy transfer (FRET). This does not occur in 5-HTP substituted amicyanin because the fluorescence quantum yield is significantly lower and the red-shift of fluorescence emission maximum decreases overlap with the near UV absorbance of copper. Characterization of the distinct fluorescence properties of 5-HTP relative to tryptophan in amicyanin provides a basis for spectroscopic interrogation of the protein microenvironment using 5-HTP, and long-distance interactions with transition metals.
Topics: 5-Hydroxytryptophan; Azurin; Bacterial Proteins; Copper; Energy Transfer; Metalloproteins; Paracoccus denitrificans; Tryptophan
PubMed: 35696758
DOI: 10.1016/j.jinorgbio.2022.111895 -
Biomolecules Sep 2019Protein-based electronics is an emerging field which has attracted considerable attention over the past decade. Here, we present a theoretical study of the formation and...
Protein-based electronics is an emerging field which has attracted considerable attention over the past decade. Here, we present a theoretical study of the formation and electronic structure of a metal-protein-metal junction based on the blue-copper azurin from pseudomonas aeruginosa. We focus on the case in which the protein is adsorbed on a gold surface and is contacted, at the opposite side, to an STM (Scanning Tunneling Microscopy) tip by spontaneous attachment. This has been simulated through a combination of molecular dynamics and density functional theory. We find that the attachment to the tip induces structural changes in the protein which, however, do not affect the overall electronic properties of the protein. Indeed, only changes in certain residues are observed, whereas the electronic structure of the Cu-centered complex remains unaltered, as does the total density of states of the whole protein.
Topics: Adsorption; Azurin; Biomechanical Phenomena; Copper; Density Functional Theory; Electron Transport; Gold; Microscopy, Scanning Tunneling; Models, Molecular; Molecular Dynamics Simulation; Protein Structure, Tertiary; Pseudomonas aeruginosa; Surface Properties
PubMed: 31546917
DOI: 10.3390/biom9090506 -
Protein Science : a Publication of the... Nov 2021We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with...
Copper nitrite reductase from Sinorhizobium meliloti 2011: Crystal structure and interaction with the physiological versus a nonmetabolically related cupredoxin-like mediator.
We report the crystal structure of the copper-containing nitrite reductase (NirK) from the Gram-negative bacterium Sinorhizobium meliloti 2011 (Sm), together with complex structural alignment and docking studies with both non-cognate and the physiologically related pseudoazurins, SmPaz1 and SmPaz2, respectively. S. meliloti is a rhizobacterium used for the formulation of Medicago sativa bionoculants, and SmNirK plays a key role in this symbiosis through the denitrification pathway. The structure of SmNirK, solved at a resolution of 2.5 Å, showed a striking resemblance with the overall structure of the well-known Class I NirKs composed of two Greek key β-barrel domains. The activity of SmNirK is ~12% of the activity reported for classical NirKs, which could be attributed to several factors such as subtle structural differences in the secondary proton channel, solvent accessibility of the substrate channel, and that the denitrifying activity has to be finely regulated within the endosymbiont. In vitro kinetics performed in homogenous and heterogeneous media showed that both SmPaz1 and SmPaz2, which are coded in different regions of the genome, donate electrons to SmNirK with similar performance. Even though the energetics of the interprotein electron transfer (ET) process is not favorable with either electron donors, adduct formation mediated by conserved residues allows minimizing the distance between the copper centers involved in the interprotein ET process.
Topics: Azurin; Bacterial Proteins; Crystallography, X-Ray; Nitrite Reductases; Protein Domains; Sinorhizobium meliloti
PubMed: 34562300
DOI: 10.1002/pro.4195 -
Proceedings of the National Academy of... Oct 2023The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His) with a backbone amine, is found in metalloproteins with diverse functions. A primary example...
The "Histidine-brace" (His-brace) copper-binding site, composed of Cu(His) with a backbone amine, is found in metalloproteins with diverse functions. A primary example is lytic polysaccharide monooxygenase (LPMO), a class of enzymes that catalyze the oxidative depolymerization of polysaccharides, providing not only an energy source for native microorganisms but also a route to more effective industrial biomass conversion. Despite its importance, how the Cu His-brace site performs this unique and challenging oxidative depolymerization reaction remains to be understood. To answer this question, we have designed a biosynthetic model of LPMO by incorporating the Cu His-brace motif into azurin, an electron transfer protein. Spectroscopic studies, including ultraviolet-visible (UV-Vis) absorption and electron paramagnetic resonance, confirm copper binding at the designed His-brace site. Moreover, the designed protein is catalytically active towards both cellulose and starch, the native substrates of LPMO, generating degraded oligosaccharides with multiturnovers by C1 oxidation. It also performs oxidative cleavage of the model substrate 4-nitrophenyl-D-glucopyranoside, achieving a turnover number ~9% of that of a native LPMO assayed under identical conditions. This work presents a rationally designed artificial metalloenzyme that acts as a structural and functional mimic of LPMO, which provides a promising system for understanding the role of the Cu His-brace site in LPMO activity and potential application in polysaccharide degradation.
Topics: Mixed Function Oxygenases; Copper; Histidine; Polysaccharides
PubMed: 37844252
DOI: 10.1073/pnas.2308286120 -
Journal of the American Chemical Society Nov 2020We observe reversible, bias-induced switching of conductance via a blue copper protein azurin mutant, , with a nearly 10-fold increase at || > 0.8 V than at lower bias....
We observe reversible, bias-induced switching of conductance via a blue copper protein azurin mutant, , with a nearly 10-fold increase at || > 0.8 V than at lower bias. No such switching is found for wild-type azurin, , up to |1.2 V|, beyond which irreversible changes occur. The mutant will, when positioned between electrodes in a solid-state Au-protein-Au junction, have an orientation opposite that of with respect to the electrodes. Current(s) via both proteins are temperature-independent, consistent with quantum mechanical tunneling as dominant transport mechanism. No noticeable difference is resolved between the two proteins in conductance and inelastic electron tunneling spectra at <|0.5 V| bias voltages. Switching behavior persists from 15 K up to room temperature. The conductance peak is consistent with the system switching in and out of resonance with the changing bias. With further input from UV photoemission measurements on Au-protein systems, these striking differences in conductance are rationalized by having the location of the Cu(II) coordination sphere in the mutant, proximal to the (larger) substrate-electrode, to which the protein is chemically bound, while for the that coordination sphere is closest to the other Au electrode, with which only physical contact is made. Our results establish the key roles that a protein's orientation and binding nature to the electrodes play in determining the electron transport tunnel barrier.
Topics: Azurin; Copper; Electrodes; Electron Transport; Gold; Mutagenesis; Photoelectron Spectroscopy; Protein Binding; Quantum Theory; Temperature
PubMed: 33141577
DOI: 10.1021/jacs.0c08836 -
Chemical Science Mar 2021Mononitrosyl and dinitrosyl iron species, such as {FeNO}, {FeNO} and {Fe(NO)}, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron...
Mononitrosyl and dinitrosyl iron species, such as {FeNO}, {FeNO} and {Fe(NO)}, have been proposed to play pivotal roles in the nitrosylation processes of nonheme iron centers in biological systems. Despite their importance, it has been difficult to capture and characterize them in the same scaffold of either native enzymes or their synthetic analogs due to the distinct structural requirements of the three species, using redox reagents compatible with biomolecules under physiological conditions. Here, we report the realization of stepwise nitrosylation of a mononuclear nonheme iron site in an engineered azurin under such conditions. Through tuning the number of nitric oxide equivalents and reaction time, controlled formation of {FeNO} and {Fe(NO)} species was achieved, and the elusive {FeNO} species was inferred by EPR spectroscopy and observed by Mössbauer spectroscopy, with complemental evidence for the conversion of {FeNO} to {Fe(NO)} species by UV-Vis, resonance Raman and FT-IR spectroscopies. The entire pathway of the nitrosylation process, Fe(ii) → {FeNO} → {FeNO} → {Fe(NO)}, has been elucidated within the same protein scaffold based on spectroscopic characterization and DFT calculations. These results not only enhance the understanding of the dinitrosyl iron complex formation process, but also shed light on the physiological roles of nitric oxide signaling mediated by nonheme iron proteins.
PubMed: 34040732
DOI: 10.1039/d1sc00364j -
Molecules (Basel, Switzerland) Jan 2024Multi-step electron transfer reactions are important to the function of many cellular systems. The ways in which such systems have evolved to direct electrons along...
Multi-step electron transfer reactions are important to the function of many cellular systems. The ways in which such systems have evolved to direct electrons along specific pathways are largely understood, but less so are the ways in which the reduction-oxidation potentials of individual redox sites are controlled. We prepared a series of three new artificial variants of azurin where a tyrosine (Tyr109) is situated between the native Cu ion and a Ru(II) photosensitizer tethered to a histidine (His107). Arginine, glutamine, or methionine were introduced as position 122, which is near to Tyr109. We investigated the rate of Cu oxidation by a flash-quench generated Ru(III) oxidant over pH values from 5 to 9. While the identity of the residue at position 122 affects some of the physical properties of Tyr109, the rates of Cu oxidation are only weakly dependent on the identity of the residue at 122. The results highlight that more work is still needed to understand how non-covalent interactions of redox active groups are affected in redox proteins.
Topics: Tyrosine; Electrons; Glutamine; Methionine; Arginine
PubMed: 38257263
DOI: 10.3390/molecules29020350