Intramolecular Electron Transfer in the Bacterial Two-Domain Multicopper Oxidase mgLAC

The kinetics of the intramolecular electron transfer process in mgLAC, a bacterial two-domain multicopper oxidase (MCO), were investigated by pulse radiolysis. The reaction is initiated by CO₂^– radicals produced in anaerobic, aqueous solutions of the enzyme by microsecond pulses of radiation. A sequence of pulses of CO₂^– radicals enables examination of the reductive half-cycle of the MCO catalysis. This is done by titrations of the Type 1 (T1) Cu­(II) site and monitoring of the time course and amplitude of its reoxidation by internal electron transfer (ET) to the Type 3 site. Comparison of the internal ET kinetics observed for mgLAC with those of other MCOs studied by pulse radiolysis shows that they exhibit distinct reactivities. One main cause for the different reactivities is the broad range of T1 copper redox potentials, from the moderate potential of bacterial enzymes to the high potential of fungal laccases, and this possibly also reflects evolutionary quaternary structural adaptation of the MCO family to the wide range of reducing substrates that they oxidize while maintaining efficient reduction of the common substrate, molecular oxygen

Doping Human Serum Albumin with Retinoate Markedly Enhances Electron Transport across the Protein

Electrons can migrate via proteins over distances that are considered long for nonconjugated systems. The nanoscale dimensions of proteins and their enormous structural and chemical flexibility makes them fascinating subjects for exploring their electron transport (ETp) capacity. One particularly attractive direction is that of tuning their ETp efficiency by “doping” them with small molecules. Here we report that binding of retinoate (RA) to human serum albumin (HSA) increases the solid-state electronic conductance of a monolayer of the protein by >2 orders of magnitude for RA/HSA ≥ 3. Temperature-dependent ETp measurements show the following with increasing RA/HSA: (a) The temperature-independent current magnitude of the low-temperature (<190 K) regime increases significantly (>300-fold), suggesting a decrease in the distance-decay constant of the process. (b) The activation energy of the thermally activated regime (>190 K) decreases from 220 meV (RA/HSA = 0) to 70 meV (RA/HSA ≥ 3)

Electron Transport via Cytochrome C on Si–H Surfaces: Roles of Fe and Heme

Monolayers of the redox protein Cytochrome C (CytC) can be electrostatically formed on an H-terminated Si substrate, if the protein- and Si-surface are prepared so as to carry opposite charges. With such monolayers we study electron transport (ETp) via CytC, using a solid-state approach with macroscopic electrodes. We have revealed that currents via holo-CytC are almost 3 orders of magnitude higher than via the heme-depleted protein (→ apo-CytC). This large difference in currents is attributed to loss of the proteins’ secondary structure upon heme removal. While removal of only the Fe ion (→ porphyrin-CytC) does not significantly change the currents via this protein at room temperature, the 30–335 K temperature dependence suggests opening of a new ETp pathway, which dominates at high temperatures (>285 K). These results suggest that the cofactor plays a major role in determining the ETp pathway(s) within CytC

Conjugated Cofactor Enables Efficient Temperature-Independent Electronic Transport Across ∼6 nm Long Halorhodopsin

We observe temperature-independent electron transport, characteristic of tunneling across a ∼6 nm thick Halorhodopsin (phR) monolayer. phR contains both retinal and a carotenoid, bacterioruberin, as cofactors, in a trimeric protein-chromophore complex. This finding is unusual because for conjugated oligo-imine molecular wires a transition from temperature-independent to -dependent electron transport, ETp, was reported at ∼4 nm wire length. In the ∼6 nm long phR, the ∼4 nm 50-carbon conjugated bacterioruberin is bound parallel to the α-helices of the peptide backbone. This places bacterioruberin’s ends proximal to the two electrodes that contact the protein; thus, coupling to these electrodes may facilitate the activation-less current across the contacts. Oxidation of bacterioruberin eliminates its conjugation, causing the ETp to become temperature dependent (>180 K). Remarkably, even elimination of the retinal-protein covalent bond, with the fully conjugated bacterioruberin still present, leads to temperature-dependent ETp (>180 K). These results suggest that ETp via phR is cooperatively affected by both retinal and bacterioruberin cofactors

Temperature-Dependent Solid-State Electron Transport through Bacteriorhodopsin: Experimental Evidence for Multiple Transport Paths through Proteins

Electron transport (ETp) across bacteriorhodopsin (bR), a natural proton pump protein, in the solid state (dry) monolayer configuration, was studied as a function of temperature. Transport changes from thermally activated at <i>T</i> > 200 K to temperature independent at <130 K, similar to what we have observed earlier for BSA and apo-azurin. The relatively large activation energy and high temperature stability leads to conditions where bR transports remarkably high current densities above room temperature. Severing the chemical bond between the protein and the retinal polyene only slightly affected the main electron transport via bR. Another thermally activated transport path opens upon retinal oxime production, instead of or in addition to the natural retinal. Transport through either or both of these paths occurs on a background of a general temperature-independent transport. These results lead us to propose a generalized mechanism for ETp across proteins, in which tunneling and hopping coexist and dominate in different temperature regimes

Solvent Accessibility in the Distal Heme Pocket of the Nitrosyl d₁‑Heme Complex of <i>Pseudomonas stutzeri</i> cd₁ Nitrite Reductase

In nitrite reductase (cd₁ NIR), the c-heme mediates electron transfer to the catalytic d₁-heme where nitrite (NO₂^–) is reduced to nitric oxide (NO). An interesting feature of this enzyme is the relative lability of the reaction product NO bound to the d₁-heme. Marked differences in the c- to d₁-heme electron-transfer rates were reported for cd₁ NIRs from different sources, such as <i>Pseudomonas stutzeri</i> (<i>P. stutzeri</i>) and <i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>). The three-dimensional structure of the <i>P. aeruginosa</i> enzyme has been determined, but that of the <i>P. stutzeri</i> enzyme is still unknown. The difference in electron transfer rates prompted a comparison of the structural properties of the d₁-heme pocket of <i>P. stutzeri</i> cd₁ NIR with those of the <i>P. aeruginosa</i> wild type enzyme (WT) and its Y10F using their nitrosyl d₁-heme complexes. We applied high field pulse electron paramagnetic resonance (EPR) techniques that detect nuclear spins in the close environment of the spin bearing Fe­(II)-NO entity. We observed similarities in the rhombic g-tensor and detected a proximal histidine ligand with ¹⁴N hyperfine and quadrupole interactions also similar to those of <i>P. aeruginosa</i> WT and Y10F mutant complexes. In contrast, we also observed significant differences in the H-bond network involving the NO ligand and a larger solvent accessibility for <i>P. stutzeri</i> attributed to the absence of this tyrosine residue. For <i>P. aeruginosa</i>, cd₁ NIR domain swapping allows Tyr₁₀ to become H-bonded to the bound NO substrate. These findings support a previous suggestion that the large difference in the c- to d₁-heme electron transfer rates between the two enzymes is related to solvent accessibility of their d₁-heme pockets

Long-Range Electron Transfer in Engineered Azurins Exhibits Marcus Inverted Region Behavior

The Marcus theory of electron transfer (ET) predicts that while the ET rate constants increase with rising driving force until it equals a reaction’s reorganization energy, at higher driving force the ET rate decreases, having reached the Marcus inverted region. While experimental evidence of the inverted region has been reported for organic and inorganic ET reactions as well as for proteins conjugated with ancillary redox moieties, evidence of the inverted region in a “protein-only” system has remained elusive. We herein provide such evidence in a series of nonderivatized proteins. These results may facilitate the design of ET centers for future applications such as advanced energy conversions

Nanoscale Electron Transport and Photodynamics Enhancement in Lipid-Depleted Bacteriorhodopsin Monomers

Potential future use of bacteriorhodopsin (bR) as a solid-state electron transport (ETp) material requires the highest possible active protein concentration. To that end we prepared stable monolayers of protein-enriched bR on a conducting HOPG substrate by lipid depletion of the native bR. The ETp properties of this construct were then investigated using conducting probe atomic force microscopy at low bias, both in the ground dark state and in the M-like intermediate configuration, formed upon excitation by green light. Photoconductance modulation was observed upon green and blue light excitation, demonstrating the potential of these monolayers as optoelectronic building blocks. To correlate protein structural changes with the observed behavior, measurements were made as a function of pressure under the AFM tip, as well as humidity. The junction conductance is reversible under pressure changes up to ∼300 MPa, but above this pressure the conductance drops irreversibly. ETp efficiency is enhanced significantly at >60% relative humidity, without changing the relative photoactivity significantly. These observations are ascribed to changes in protein conformation and flexibility and suggest that improved electron transport pathways can be generated through formation of a hydrogen-bonding network

Electronic Transport via Homopeptides: The Role of Side Chains and Secondary Structure

Many novel applications in bioelectronics rely on the interaction between biomolecules and electronically conducting substrates. However, crucial knowledge about the relation between electronic transport via peptides and their amino-acid composition is still absent. Here, we report results of electronic transport measurements via several homopeptides as a function of their structural properties and temperature. We demonstrate that the conduction through the peptide depends on its length and secondary structure as well as on the nature of the constituent amino acid and charge of its residue. We support our experimental observations with high-level electronic structure calculations and suggest off-resonance tunneling as the dominant conduction mechanism via extended peptides. Our findings indicate that both peptide composition and structure can affect the efficiency of electronic transport across peptides