Metalloprotein Electron Transfer Research Articles

Peptides and metal ions: A successful marriage for developing artificial metalloproteins.

The mutual relationship between peptides and metal ions enables metalloproteins to have crucial roles in biological systems, including structural, sensing, electron transport, and catalytic functions. The effort to reproduce or/and enhance these roles, or even to create unprecedented functions, is the focus of protein design, the first step toward the comprehension of the complex machinery of nature. Nowadays, protein design allows the building of sophisticated scaffolds, with novel functions and exceptional stability. Recent progress in metalloprotein design has led to the building of peptides/proteins capable of orchestrating the desired functions of different metal cofactors. The structural diversity of peptides allows proper selection of first- and second-shell ligands, as well as long-range electrostatic and hydrophobic interactions, which represent precious tools for tuning metal properties. The scope of this review is to discuss the construction of metal sites in de novo designed and miniaturized scaffolds. Selected examples of mono-, di-, and multi-nuclear binding sites, from the last 20 years will be described in an effort to highlight key artificial models of catalytic or electron-transfer metalloproteins. The authors' goal is to make readers feel like guests at the marriage between peptides and metal ions while offering sources of inspiration for future architects of innovative, artificial metalloproteins.

Electrochemical characterization of mutant forms of rubredoxin B from Mycobacterium tuberculosis

Electron transfer in metalloproteins is a driving force for many biological processes and widely distributed in nature. Rubredoxin B (RubB) from Mycobacterium tuberculosis is a first example among [1Fe-0S] proteins that support catalytic activity of terminal sterol-monooxygenases enabling its application in metabolic engineering. To explore the tolerance of RubB to the specific amino acid changes we evaluated the effect of surface mutations on its electrochemical properties. Based on the RubB fold we also designed the mutant with a putative additional site for protein-protein interactions to further evaluate electron transfer and electrochemical properties. The investigation of redox properties of mutant variants of RubB was done using screen-printed graphite electrodes (SPEs) modified with stable dispersion of multi-walled carbon nanotubes (MWCNTs). The redox potentials (midpoint potentials, E0Ꞌ) of mutants did not significantly differ from the wild type protein and vary in the range of −264 to −231 mV vs. Ag/AgCl electrode. However, all mutations affect electron transfer rate between the protein and electrode. Notably, the modulation of the protein-protein interactions was observed for the insertion mutant suggesting the possibility of tailoring of rubredoxin for the selected redox-partner. Overall, RubB is tolerant to the significant modifications in its structure enabling rational engineering of novel redox proteins.

Dynamical effects in metalloprotein heterogeneous electron transfer

In this work we assess the influence of physiological viscosities on metalloprotein electron transfer reactions. To that end we investigated the direct electrochemistry of the copper proteins azurin and CuA adsorbed on SAM-coated electrodes. The experimental results show a change of ET regime from non-adiabatic to friction control upon shortening tunneling distances, which is paralleled by a sigmoidal increase of the apparent ET reorganization energies. These data could be accurately described using novel Matyushov’s model, thereby validating this recent theoretical development, which however did not describe viscosity effects in its original formulation. We demonstrate that ET rate constants and effective relaxation times vary with viscosity following power laws. Moreover, the crossover parameter g that determines the ET regime could be redefined in terms of the exponents of these power laws. The magnitude of g, i.e. the extent of frictional control, was found to be protein specific as it is determined by the dynamical features of the protein milieu. Interestingly, Stokes shift and diffusional relaxation times were found to be of similar magnitude, thus resulting in non-negligible frictional control even at tunneling distances as long as 19 Å, and this effect is amplified by physiologically high viscosities, thus highlighting the influence of intracellular macromolecular crowding in modulating protein ET reactions.

Metallated Schiff-Base Macromolecules as Alternative Metalloprotein Electron Transfer Intermediates

In the construction of biosensors, enzymes function as mediators converting biological signals generated by specific biological processes, into electrochemical signals. The ideology of bio-sensor design is retention of electron transfer activity of the enzyme utilizing superior interfacial architecture. In this work a Schiff-base macromolecule has been synthesized by reflux of 2, 3-diaminonaphthalene and pyrrole-2-carboxaldehyde starting materials. The Schiff-base ligand was subsequently complexed with FeCl2?4H2O under reflux, to produce the Fe-Schiff-base complex. The Schiff-base ligand and Fe-Schiff-base complex were characterized using nuclear magnetic resonance (NMR) spectroscopy, Ultra Violet/Visible (UV/Vis) spectroscopy, Fourier transfer infrared resonance (FTIR) and electron energy loss spectroscopy (EELS) to confirm the structure of the synthesis products. NMR spectroscopy confirmed the imide linkage of Schiff-base formation as two symmetrical peaks at 8.1 and 7.7 ppm respectively. Comparison of starting materials and product spectra by UV/Vis spectroscopy confirmed the disappearance of the diaminonaphthalene peak at 250 nm as evidence of complete conversion to product. FTIR spectroscopy of the Schiff-base ligand confirmed the formation of the imine bond at 1595 cm-1. EELS spectra comparing FeCl2?4H2O and the Fe-Schiff-base complex, showed good agreement in the energy loss profiles associated with changes to the electronic arrangement of Fe d-orbitals. EDS clearly identified a spectral band for Fe (7 - 8 eV) in the Fe-Schiff-base complex. Electrochemical evaluation of the Fe-Schiff-base complex was compared to the electrochemical signature of denatured cytochrome-C using cyclic voltammetry and square wave voltammetry. The Fe2+/Fe3+ quasi-reversible behavior for iron in the metallated complex was observed at -0.430 V vs. Ag/AgCl, which is consistent with reference values for iron in macromolecular structures.

The role of molecular crowding in long-range metalloprotein electron transfer: Dissection into site- and scaffold-specific contributions

Here we report the effect of molecular crowding on long-range protein electron transfer (ET) and disentangle the specific responses of the redox site and the protein milieu. To this end, we studied two different one-electron redox proteins that share the cupredoxin fold but differ in the metal center, T1 mononuclear blue copper and binuclear CuA, and generated chimeras with hybrid properties by incorporating different T1 centers within the CuA scaffold or by swapping loops between orthologous proteins from different organisms to perturb the CuA site. The heterogeneous ET kinetics of the different proteins was studied by protein film electrochemistry at variable electronic couplings and in the presence of two different crowding agents. The results reveal a strong frictional control of the ET reactions, which for 10 Å tunnelling distances results in a 90% drop of the ET rate when viscosity is matched to that of the mitochondrial interior (ca. 55 cP) by addition of either crowding agent. The effect is ascribed to the dynamical coupling of the metal site and the milieu, which for T1 is found to be twice stronger than for CuA, and the activation energy of protein-solvent motion that is dictated by the overall scaffold. This work highlights the need of explicitly considering molecular crowding effects in protein ET.

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer.

Determining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pK a values of multiple non‐coordinating residues (predominantly histidine) that involve between 0.42–0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein–cofactor–solvent complex.

Direct Measurement of Charge Regulation in Metalloprotein Electron Transfer

AbstractDetermining whether a protein regulates its net electrostatic charge during electron transfer (ET) will deepen our mechanistic understanding of how polypeptides tune rates and free energies of ET (e.g., by affecting reorganization energy, and/or redox potential). Charge regulation during ET has never been measured for proteins because few tools exist to measure the net charge of a folded protein in solution at different oxidation states. Herein, we used a niche analytical tool (protein charge ladders analyzed with capillary electrophoresis) to determine that the net charges of myoglobin, cytochrome c, and azurin change by 0.62±0.06, 1.19±0.02, and 0.51±0.04 units upon single ET. Computational analysis predicts that these fluctuations in charge arise from changes in the pKa values of multiple non‐coordinating residues (predominantly histidine) that involve between 0.42–0.90 eV. These results suggest that ionizable residues can tune the reactivity of redox centers by regulating the net charge of the entire protein–cofactor–solvent complex.

Approaches to single-molecule studies of metalloprotein electron transfer using scanning probe-based techniques.

The single-molecule properties of metalloproteins have provided an intensely active research area in recent years. This brief review covers some of the techniques used to prepare, measure and analyse the electron transfer properties of metalloproteins, concentrating on scanning tunnelling microscopy-based techniques and advances in attachment of proteins to electrodes.

Simulation insight into the cytochrome c adsorption on graphene and graphene oxide surfaces

Abstract Graphene-based materials might serve as an ideal platform for the regulation and promotion of metalloprotein electron transfer (ET); however, the underpinning mechanism at the molecular level has not yet been fully revealed. The orientation of cytochrome c (Cyt c) on surfaces is vital for ET. In this work, the orientation of Cyt c on graphene and graphene oxide (GO) surfaces, as well as the dominant driving forces, the conformational change and the ET pathways were investigated by molecular dynamics simulations. The results show that Cyt c is adsorbed onto the GO surface mainly through lysine residues; whereas hydrophobic interaction contributes to the Cyt c adsorption on graphene surface. There is no significant conformational change of Cyt c upon adsorption. The heme plane of Cyt c tends to be horizontally oriented and far away from the graphene surface, which is not conducive to ET. On the GO surface, the heme plane is slightly deviated from the normal direction to the surface and the axial ligand Met80 is much closer to the surface, which facilitates the ET. These findings shed some light on the ET mechanism of Cyt c on graphene-based materials and provide guidance for the development of bionic electronic devices.

The power of three.

Last month, the Executive Committee of the International Union of Crystallography approved the appointment of Dr Zbigniew Dauter as joint Section Editor of Acta Crystallographica Section F: Structural Biology Communications. This is great news for our journal, our authors and readers. Zbyszek is renowned in the field of macromolecular crystallography. He has been active in crystallography for more than 40 years. He started out investigating structures of small organic compounds with potential medical applications and obtained his PhD in 1975 from the Technical University of Gdansk, Poland, for the dissertation titled The structure of 1-nitro-9-(3′-dimethyl-aminopropylamino) acridine (C283). In time his scientific interests shifted to macromolecular crystallography and he has since been involved in the structure elucidation of over 70 proteins, among them electron-transfer metalloproteins such as rubredoxin and ferredoxin; proteinases from the subtilisin and trypsin families and their inhibitors; various other enzymes such as dUTPases, dehydrogenases, glycosyl hydrolases, aldolases, HIV reverse transcriptase; and larger complexes such as photosystem II or carnation mottle virus. In addition to these achievements, Zbyszek is probably best known for his seminal contributions to the optimization of diffraction data collection especially using synchrotron radiation. He has published several texts on this topic, presented numerous lectures and seminars, and served as a tutor in many, many data collection workshops. He participated in the development of the protocols for analysis and validation of protein structures at atomic resolution, and he is investigating how the weak resonant signal from anomalous scatterers, present or introduced into crystals of macromolecules, can be utilized most effectively for phasing purposes. He is the inventor of the quick soaking method using halides (bromide and iodide), a method which is now widely known as dauterization of macromolecular crystals. More recently, he has developed an interest in exploiting radiation-damage signals for the phasing of macromolecular structures. In 2010, his work was recognised when he was awarded the highest science award from the Polish Academy of Sciences. In total, Zbyszek has authored or co-authored about 330 papers in international journals and he is one of only a few crystallographers who have published in all six Acta journals. Up to now he has published three papers in Acta A, eight in Acta B, 15 in Acta C, 102 in Acta D, eight in Acta E and eight in Acta F. He has been a Section Editor of Acta D from 2003 until 2015 and we are very fortunate that he has agreed to join us now as a Section Editor of Acta F. We welcome him to our team very warmly. Let’s keep up the good work, Zbyszek!

Probing the nature of electron transfer in metalloproteins on graphene-family materials as nanobiocatalytic scaffold using electrochemistry

Graphene-based nanomaterials have shown great promise not only in nanoelectronics due to ultrahigh electron mobility but also as biocatalytic scaffolds owing to irreversible protein surface adsorption and facilitating direct electron transfer. In this work, we synthesized stable dispersions of graphene using liquid-phase exfoliation approach based on non-covalent interactions between graphene and 1-pyrenesulfonic acid sodium salt (Py–1SO3), 1-pyrenemethylamine salt (Py − Me-NH2) and Pluronic® P-123 surfactant using only water as solvent compatible with biomolecules. The resulting graphene nanoplatelets (Gr_LPE) are characterized by a combination of analytical (microscopy and spectroscopy) techniques revealing mono- to few-layer graphene displaying that the exfoliation efficiency strongly depends upon the type of pyrene-based salts and organic surfactants. Moreover being completely water-based approach, we build robust nanoscaffolds of graphene-family nanomaterials (GFNs) namely, monolayer graphene, Gr_LPE (the one prepared with Pluronic® P-123), graphene oxide (GO) and its reduced form (rGO) on glassy carbon electrode surface with three important metalloproteins include cytochrome c (Cyt c) [for electron transfer], myoglobin (Mb) [for oxygen storage] and horseradish peroxidase (HRP) [for catalyzing the biochemical reaction]. In order to demonstrate the nanobiocatalytical activity of these proteins, we used electrochemical interfacial direct electron transfer (DET) kinetics and attempt to determine the rate constant (kET) using two different analytical approaches namely, linear sweep voltammetry and Laviron’s theory. We elucidated that all of the metalloproteins retain their structural integrity (secondary structure) upon forming mixtures with GFNs confirmed through optical and vibrational spectroscopy and biological activity using electrochemistry. Among the GFNs studied, Gr-LPE, GO and rGO support the efficient electrical wiring of the redox centers (with an increase in catalytic efficiency of Cyt c and Mb in the presence of GFNs attributed partially to the surface functional (carboxyl, epoxide and hydroxyl) groups on GO and rGO facilitating rapid charge transfer.

Axial interactions in the mixed-valent Cu A active site and role of the axial methionine in electron transfer

Within Cu-containing electron transfer active sites, the role of the axial ligand in type 1 sites is well defined, yet its role in the binuclear mixed-valent CuA sites is less clear. Recently, the mutation of the axial Met to Leu in a CuA site engineered into azurin (CuA Az) was found to have a limited effect on E(0) relative to this mutation in blue copper (BC). Detailed low-temperature absorption and magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance studies on CuA Az (WT) and its M123X (X = Q, L, H) axial ligand variants indicated stronger axial ligation in M123L/H. Spectroscopically validated density functional theory calculations show that the smaller ΔE(0) is attributed to H2O coordination to the Cu center in the M123L mutant in CuA but not in the equivalent BC variant. The comparable stabilization energy of the oxidized over the reduced state in CuA and BC (CuA ∼ 180 mV; BC ∼ 250 mV) indicates that the S(Met) influences E(0) similarly in both. Electron delocalization over two Cu centers in CuA was found to minimize the Jahn-Teller distortion induced by the axial Met ligand and lower the inner-sphere reorganization energy. The Cu-S(Met) bond in oxidized CuA is weak (5.2 kcal/mol) but energetically similar to that of BC, which demonstrates that the protein matrix also serves an entatic role in keeping the Met bound to the active site to tune down E(0) while maintaining a low reorganization energy required for rapid electron transfer under physiological conditions.

Effects of quantum coherence in metalloprotein electron transfer

Many intramolecular electron transfer (ET) reactions in biology are mediated by metal centers in proteins. This process is commonly described by a model of diffusive hopping according to the semiclassical theories of Marcus and Hopfield. However, recent studies have raised the possibility that nontrivial quantum mechanical effects play a functioning role in certain biomolecular processes. Here, we investigate the potential effects of quantum coherence in biological ET by extending the semiclassical model to allow for the possibility of quantum coherent phenomena using a quantum master equation based on the Holstein Hamiltonian. We test the model on the structurally defined chain of seven iron-sulfur clusters in nicotinamide adenine dinucleotide plus hydrogen:ubiquinone oxidoreductase (complex I), a crucial respiratory enzyme and one of the longest chains of metal centers in biology. Using experimental parameters where possible, we find that, in limited circumstances, a small quantum mechanical contribution can provide a marked increase in the ET rate above the semiclassical diffusive-hopping rate. Under typical biological conditions, our model reduces to well-known diffusive behavior.

The Role of Hemoglobin's C-terminal Region: the Work of Eraldo Antonini

Studies on Carboxypeptidase Digests of Human Hemoglobin (Antonini, E., Wyman, J., Zito, R., Rossi-Fanelli, A., and Caputo, A. (1961) J. Biol. Chem. 236, PC60–PC63) The Effect of Oxygenation on the Rate of Digestion of Human Hemoglobins by Carboxypeptidases (Zito, R., Antonini, E., and Wyman, J. (1964) J. Biol. Chem. 239, 1804–1808)

Surface-induced intramolecular electron transfer in multi-centre redox metalloproteins: the di-haem proteincytochrome c4 in homogeneous solution and at electrochemical surfaces

Intramolecular electron transfer (ET) between transition metal centres is a core featureof biological ET and redox enzyme function. The number of microscopic redoxpotentials and ET rate constants is, however, mostly prohibitive for experimentalmapping, but two-centre proteins offer simple enough communication networksfor complete mapping to be within reach. At the same time, multi-centre redoxproteins operate in a membrane environment where conformational dynamics andET patterns are quite different from the conditions in a homogeneous solution.The bacterial respiratory di-haem protein Pseudomonas stutzeri cytochromec4 offers a prototype target for environmental gating of intra-haem ET. ET between P. stutzeri cytc4 and small molecular reaction partners in solution appears completely dominated byintermolecular ET of each haem group/protein domain, with no competing intra-haem ET,for which accompanying propionate-mediated proton transfer is a further barrier. Theprotein can, however, be immobilized on single-crystal, modified Au(111) electrode surfaceswith either the low-potential N terminal or the high-potential C terminal domain facing thesurface, clearly with fast intramolecular ET as a key feature in the electrochemical two-ETprocess. This dual behaviour suggests a pattern for multi-centre redox metalloproteinfunction. In a homogeneous solution, which is not the natural environment of cytc4, the two haem group domains operate largely independently with conformationsprohibitive for intramolecular ET. Binding to a membrane or electrochemicalsurface, however, triggers conformational opening of intramolecular ET channels.The haem group orientation in P. stutzeri cytc4 is finally noted to offer a case for orientation dependent electronic rectification between asubstrate and a tip in electrochemical in situ scanning tunnelling microscopy or nanoscaleelectrode configurations.

Unravelling single metalloprotein electron transfer by scanning probe techniques

This review is intended to account for the experimental and theoretical achievements obtained in a period of about 15 years on the investigation of the electron transport through single redox metalloproteins by scanning probe techniques. A highly focussed research effort has been deployed by the scientists active in this particular field towards measuring and interpreting electronic current signals flowing via blue copper, redox metalloproteins (e.g. azurin). The field has taken a remarkable advantage of the use of electrochemically assisted scanning tunnelling microscope (EC-STM) which has allowed to probe single molecule signals under full control of all the potential values involved in the experiments. This experimental activity has both triggered more comprehensive theoretical interpretations and has been, in its turn, stimulated by theoreticians to test always new predictions. The authors hope to have succeeded in providing the reader with a valuable appraisal of this fascinating field.

The 2004 Benjamin Franklin Medal in Chemistry presented to Harry B. Gray

The Franklin Institute, Philadelphia, Pennsylvania, awarded the 2004 Benjamin Franklin Medal in Chemistry to Harry B. Gray for his pioneering contributions in the field of electron transfer in metalloproteins. In a series of elegant and challenging experiments beginning in the late 70s, Gray and his coworkers have shown that the transfer of electrons in metalloproteins can proceed over long distances (~ 20 Å) and at fast rates. These experiments have involved the regiospecific functionalization of structurally characterized electron transfer proteins with ruthenium complexes, coupled with laser excitation and transient spectroscopy. Probing the effects of thermodynamic driving force, temperature, donor–acceptor distance and electronic coupling, Gray has shaped our detailed current understanding of the principles governing biological electron flow. The mechanism of electron transfer has been identified as electron tunneling mediated by the molecules separating donor and acceptor. Tunneling timetables have been established for various intervening media. Important biological processes like respiration and photosynthesis depend on facile electron transfer, and Gray's contribution serves as the fundamental basis for understanding these and many related reactions.

Electron Transfer and Redox Metalloenzyme Catalysis at the Single‐Molecule Level

AbstractVoltammetry based on single‐crystal, atomically‐planar metal electrodes is novel in bioelectrochemistry. Together with in situ scanning tunneling microscopy (STM) directly in aqueous buffer, single‐crystal voltammetry has disclosed new detail in molecular adsorption and interfacial electron transfer (ET). Image interpretation requires, however, theoretical support, as STM represents both electronic and topographic features. Molecules with accessible redox levels offer other insight into electron tunneling mechanisms, addressed in detail for ET metalloproteins. We present here in situ STM of the blue redox metalloenzyme copper nitrite reductase (Achromobacter xylosoxidans, Ax CuNiR) on Au(111) electrode surfaces modified by a self‐assembled cysteamine monolayer. Ax CuNiR displays strong nitrite reduction waves in this environment. Ax CuNiR/cysteamine/Au(111) surfaces were imaged at KNO2 concentrations where most of the enzyme is in the enzyme‐substrate bound state.Molecular resolution for both cysteamine/Au(111) and Ax CuNiR/cysteamine/Au(111) electrode surfaces was achieved. The enzyme coverage is about 1.5 × 10−13 mol cm−2, which is low compared with an ideal close‐packed monolayer. The adlayer behaves as an assembly of individual molecules, reflected in distributions of molecular appearance, although a number of molecules do show the triangular shape of the trimeric Ax CuNiR structure. The apparent average molecular height is about 11 Å. This suggests that details of electronic structures and larger assemblies are needed to disentangle enzyme mechanisms at the single‐molecule level.

Genetic modulation of metalloprotein electron transfer at bare gold.

Engineered metalloproteins and enzymes can be self assembled on pristine gold electrodes in robust, electrochemically-addressable, arrays.

Intervalence electron transfer through a thiolate bridge ligand: a Fe III–S–R–Fe II mixed valence complex

The reaction of Cp(dppe)FeI with the ligands 2,2′- and 4,4′-dithiobispyridine (S 2(Py) 2) give the mononuclear or binuclear complexes of the type [Cp(dppe)Fe-S 2(Py) 2]PF 6, [Cp(dppe)FeSPy]PF 6 or [{Cp(dppe)Fe} 2-μ-SPy](PF 6) 2 depending on the reaction condition. Reaction of Cp(dppe)FeI with dithiobispyridines in presence of TlPF 6 as halide abstractor and using CH 2Cl 2 as a solvent gives the complexes [Cp(dppe)Fe-4,4′-S 2(Py) 2) 2]PF 6 ( 1) and [CpFe(dppe)-2,2′-S 2(Py) 2]PF 6 ( 2) whereas the same reaction using CH 3OH as a solvent and NH 4PF 6 as the halide abstractor leads to the formation of the Fe III–thiolate complex [Cp(dppe)Fe-2,2′-SPy]PF 6 ( 3) and the mixed-valence complex [Cp(dppe)Fe III-μSPy-Fe II(dppe)Cp](PF 6) 2 ( 4). Magnetic and ESR measurements are in agreement with one unpaired electron delocalized between them. Mössbauer data indicate clearly the presence of two different iron sites, each one of the N-bonded and S-bonded iron atoms, with intermediate oxidation state Fe IIFe III. An electron transfer intervalence absorption was observed for this complex at 780 nm (in CH 2Cl 2). By applying the Hush theory the intervalence parameters were obtained; α=0.028, H ab=361 cm −1 which indicate Class II Robin–Day. Estimation of the rate electron transfer affords a value k th=6.5×10 6 s −1. Solvent effect on the intervalence transition follow the Hush prediction for high dielectric constants solvents which permit the evaluation of the outer and inner-sphere reorganizational parameters, which were analyzed and discussed. The electronic interaction parameters compare well with those found for electron transfer in metalloproteins.