Tuning the rate and pH accessibility of a conformational electron transfer gate

Methods to fine-tune the rate of a fast conformational electron transfer (ET) gate involving a His-heme alkaline conformer of iso-1-cytochrome c (iso-1-Cytc) and to adjust the pH accessibility of a slow ET gate involving a Lys-heme alkaline conformer are described. Fine-tuning the fast ET gate employs a strategy of making surface mutations in a substructure unfolded in the alkaline conformer. To make the slow ET gate accessible at neutral pH, the strategy involves mutations at buried sequence positions which are expected to more strongly perturb the stability of native versus alkaline iso-1-Cytc. To fine-tune the rate of the fast His 73-heme ET gate, we mutate the surface-exposed Lys 79 to Ala (A79H73 variant). This mutation also simplifies ET gating by removing Lys 79, which can serve as a ligand in the alkaline conformer of iso-1-Cytc. To adjust the pH accessibility of the slow Lys 73-heme ET gate, we convert the buried side chain Asn 52 to Gly and also mutate Lys 79 to Ala to simplify ET gating (A79G52 variant). ET kinetics is studied as a function of pH using hexaammineruthenium(II) chloride (a6Ru2+) to reduce the variants. Both variants show fast direct ET reactions dependent on [a6Ru2+] and slower gated ET reactions that are independent of [a6Ru2+]. The observed gated ET rates correlate well with rates for the alkaline-to-native state conformational change measured independently. Together with the previously reported H73 variant (Baddam, S.; Bowler, B. E. J. Am. Chem. Soc. 2005, 127, 9702-9703), the A79H73 variant allows His 73-heme-mediated ET gating to be fine-tuned from 75 to 200 ms. The slower Lys 73-heme (15-20 s time scale) ET gate for the A79G52 variant is now accessible over the pH range 6-8.     

A voltammetric study of interdomain electron transfer within sulfite oxidase

Protein film voltammetry of chicken liver sulfite oxidase (SO) bound at the pyrolytic graphite "edge" or modified gold electrodes shows that catalytic electron transport is controlled by the inherent electrochemical characteristics of the heme b domain and conformational changes that allow intramolecular electron transfer with the molybdenum active site. In the absence of sulfite, a single nonturnover electrochemical signal is observed at +90 mV (vs SHE) that is assigned to heme b. In the presence of sulfite, this signal transforms into a catalytic wave at similar potential. The shape and negligible pH dependence of this wave indicate that catalytic turnover is controlled by the one-electron transfers through heme b. The smaller turnover numbers obtained in this experiment (k(cat) approximately 2-4 s(-1), as compared to 100 s(-1) in solution) suggest that only a small fraction of SO is bound at the electrode in a manner that permits the conformational change necessary for fast interdomain electron transfer.     

Ultrafast Heme Dynamics of Ferric Cytochrome c in Different Environments: Electronic,Vibrational, and Conformational Relaxation

The excited‐state dynamics of ferric cytochrome c (Cyt c), an important electron‐transfer heme protein, in acidic to alkaline medium and in its unfolded form are investigated by using femtosecond pump–probe spectroscopy, exciting the heme and Tryptophan (Trp) to understand the electronic, vibrational, and conformational relaxation of the heme. At 390 nm excitation, the electronic relaxation of heme is found to be ≈150 fs at different pH values, increasing to 480 fs in the unfolded form. Multistep vibrational relaxation dynamics of the heme, including fast and slow processes, are observed at pH 7. However, in the unfolded form and at pH 2 and 11, fast phases of vibrational relaxation dominate, revealing the energy dissipation occurring through the covalent bond interaction between the heme and the nearest amino acids. A significant shortening of the excited‐state lifetime of Trp is observed at various pH values at 280 nm excitation due to resonance energy transfer to the heme. The longer time constant (25 ps) observed in the unfolded form is attributed to a complete global conformational relaxation of Cyt c.     

Direct and mediated electron transfer catalyzed by anionic tobacco peroxidase

The properties of anionic tobacco peroxidase (TOP) adsorbed on graphite electrode have been studied in direct and mediated electron transfer in a wall-jet flow injection system. The percentage of tobacco peroxidase molecules active in directelectron transfer is about 83%, which is higher than that for horeradish peroxidase (40–50%). This observation is explained in terms of the lower degree of glycosylation of TOP compared with horseradish peroxidase and, therefore, a reduced in terference from the oligosaccharide chains with direct electron transfer. Calcium ions cause an 11% drop in the reaction rate constant toward hydrogen peroxide. The detection limit of calcium chloride has been estimated as 5 m M. The results obtained by means of bioeletrochemistry, stopped-flow kinetics, and structural modeling provide evidence for the interaction between calcium cations and negatively charged residues at the distal domain (Glu-141, heme propionates, Asp-79, Asp-80) blocking the activesite. The observation that both soluble and immobilized enzyme under go conformational changes resulting in the blockade of the active site indicates that the immobilized enzyme preserves conformational flexibility. An even stronger suppressing effect of calcium ions on the rate constant for mediated electron transfer was observed. In the case of direct electron transfer, this couldmean that there is nodirect contact between the electrode and the active site of TOP. The electrons are shuttled from the active site to the surface of the electrode through electron transfer pathways in the protein globule that are sensitive to protein conformational changes.     

Reorganization of immobilized horse and yeast cytochrome c induced by pH changes or nitric oxide binding

The redox properties of horse and yeast cytochrome c electrostatically immobilized on carboxylic acid-terminated self-assembled monolayers (SAMs) have been determined over a broad pH range (pH 3.5-8) in the absence and presence of nitric oxide. Below pH 6, both proteins exhibit comparable midpoint potentials, coverages, and electron-transfer rate constants, which suggests that they are adsorbed on the SAM in a similar fashion. Above pH 6, a sharp decrease in electron-transfer rate constants is observed for immobilized yeast cytochrome c, which is indicative of a change in the electron tunneling pathway between the heme and the electrode and hence suggests that the protein reorients on the surface. Such a decrease is not observed for horse cytochrome c and therefore must be related to the specific charge distribution on yeast cytochrome c. Apart from the charge distribution on the protein, the reorientation also seems to be related to the charge on the SAM surface. The presence of nitric oxide causes a decrease in electron-transfer rate constants of both yeast and horse cytochrome c at low pH. This is probably due to the fact that nitric oxide induces a conformational change of the protein and also changes the reorganization energy for electron transfer.     

Redox processes of cytochrome c immobilized on solid supported polyelectrolyte multilayers

The heme protein cytochrome c (Cyt-c), immobilized on polyelectrolyte multilayers on a silver electrode, was studied by stationary and time-resolved surface-enhanced resonance Raman (SERR) spectroscopy to probe the redox site structure and the mechanism and dynamics of the potential-dependent interfacial processes. The layers were built up by sequential adsorption of polycations (poly[ethylene imine] (PEI); polyallylamine hydrochloride (PAH)) and polyanions (poly[styrene sulfonate] (PSS)). All multilayers terminated by PSS electrostatically bind Cyt-c. On PEI/PSS coatings, Cyt-c is peripherally bound and fully redox-active. Due to the interfacial potential drop, the apparent redox potential is lowered by 40 mV compared to that in solution. The rate constant for the heterogeneous electron transfer (ET) of ca. 0.1 s(-1) is consistent with electron tunneling through largely ordered PEI/PSS layers. ET is coupled to a reversible conformational transition of Cyt-c that involves a change of the coordination pattern of the heme. Additional (PAH/PSS) double layers cause a broadening of the redox transition and a drastic negative shift of the redox potential, which is attributed to the formation of PSS/Cyt-c complexes. It is concluded that Cyt-c can effectively compete with PAH for binding of PSS, resulting in a rearrangement of the layered structure and a penetration of the PSS-bound Cyt-c into the PAH/PSS double layers. This conclusion is consistent with SERR intensity and quartz microbalance measurements. ET was found to be overpotential-independent and faster than that for PEI/PSS coatings, which is interpreted in terms of specific PSS/Cyt-c complexes serving as gates for the heterogeneous ET.     

Electrochemistry of P450cin: new insights into P450 electron transfer

Electrochemistry of bacterial cytochrome P450cin (CYP176A) reveals that, unusually, substrate binding does not affect the heme redox potential, although a marked pH dependence is consistent with a coupled single electron/single proton transfer reaction in the range 6 < pH < 10.     

Catalytic reduction of dioxygen and nitrite ion at a Met80Ala cytochrome c-functionalized electrode

The Met80Ala variant of yeast iso-1-cytochrome c, immobilized on a gold electrode, is found to exchange electrons efficiently with it in nondenaturing conditions and to provide robust and persistent catalytic currents for O 2 and nitrite ion reduction from pH 3 to 11. Direct covalent protein linkage to gold yields the best electrochemical and electrocatalytic performances without drastically affecting the structural properties of the bound protein compared to the freely diffusing species. Therefore, this biocatalytic interface can be of use for the amperometric detection of the above species, which are of great environmental, industrial, and clinical interest, with particular reference to the exploitation in nanostructured biosensing devices. This work shows that the use of a small engineered electron transfer (ET) protein, featuring an axial heme iron coordination position available for the binding of exogenous ligands, in place of a large heme enzyme is a viable strategy for the improvement of the heterogeneous ET rate and the stability and efficiency of sensing gold-protein interfaces over a wide range of T and pH.     

Excitation wavelength dependence of primary charge separation in reaction centers from Rhodobacter sphaeroides

The excitation wavelength dependence of the initial electron transfer rate in both wild type and mutant reaction centers from Rhodobacter sphaeroides has been studied between 840 and 920 nm as a function of temperature (10-295 K). The dynamics of primary charge separation show no resolvable excitation wavelength dependence at room temperature over this spectral range. A small variation in rate with excitation wavelength is observed at cryogenic temperatures. The low temperature results cannot be explained in terms either of a nonequilibrium model that assumes that the primary charge separation starts from a vibrationally hot state or a model that assumes a static inhomogeneous distribution of electron transfer driving forces. Instead these results are consistent with the concept that primary charge separation kinetics are controlled by the dynamics of protein conformational diffusion.     

Redox and redox-coupled processes of heme proteins and enzymes at electrochemical interfaces

Modern bioelectrochemical methods rely upon the immobilisation of redox proteins and enzymes on electrodes coated with biocompatible materials to prevent denaturation. However, even when protein denaturation is effectively avoided, heterogeneous protein electron transfer is often coupled to non-Faradaic processes like reorientation, conformational transitions or acid-base equilibria. Disentangling these processes requires methods capable of probing simultaneously the structure and reaction dynamics of the adsorbed species. Here we provide an overview of the recent developments in Raman and infrared surface-enhanced spectroelectrochemical techniques applied to the study of soluble and membrane bound redox heme proteins and enzymes. Possible biological implications of the findings are critically discussed.     

Design of a five-coordinate heme protein maquette: a spectroscopic model of deoxymyoglobin

The substitution of 1-methyl-l-histidine for the histidine heme ligands in a de novo designed four-alpha-helix bundle scaffold results in conversion of a six-coordinate cytochrome maquette into a self-assembled five-coordinate mono-(1-methyl-histidine)-ligated heme as an initial maquette for the dioxygen carrier protein myoglobin. UV-vis, magnetic circular dichroism, and resonance Raman spectroscopies demonstrate the presence of five-coordinate mono-(1-methyl-histidine) ligated ferrous heme spectroscopically similar to deoxymyoglobin. Thermodynamic analysis of the ferric and ferrous heme dissociation constants indicates greater destabilization of the ferric state than the ferrous state. The ferrous heme protein reacts with carbon monoxide to form a (1-methyl-histidine)-Fe(II)(heme)-CO complex; however, reaction with dioxygen leads to autoxidation and ferric heme dissociation. These results indicate that negative protein design can be used to generate a five-coordinate heme within a maquette scaffold.     

Photochemical initiation of electron transfer reactions.

It is quite apparent that the use of photoinitiated electron transfer has become a powerful, if not dominating, technique in the study of biological electron transfer. It provides a means to measure directly very fast processes and, through the choice of approach (flavin semiquinones or related, metal substitution in hemes or modification with ruthenium) and experimental conditions, provides the ability to probe different features of the electron transfer mechanism. Nevertheless, much remains to be done to fully understand biological electron transfer. The use of photoinitiated electron transfer has clearly established a role for a number of factors involved in controlling the kinetics of electron transfer, including driving force, distance, intervening media, dynamics (conformational gating) and orientation of redox centers. However, we have only scratched the surface in regard to understanding in molecular terms the details of electron transfer in physiologically relevant systems. Thus, even relatively simple and well characterized systems like cytochrome c-cytochrome c peroxidase remain obscure in terms of the through-protein electron paths (intervening media) and the role of protein dynamics in controlling electron transfer kinetics. Indeed, it is the through-protein paths and conformational gating that are unique to biological systems and provide nature with the capability of modulating electron transfer kinetics to optimize biological function. Of the techniques described here, the use of flavin semiquinones is clearly the least invasive in that there is no evidence that flavin semiquinones bind to or perturb physiologically relevant systems. However, this approach is constrained in that precise distances and orientations are not always known, and the range of driving forces available is limited. In contrast, metal substitution and ruthenation allow the positions of interacting redox centers to be reasonably well defined and can provide a very large range of driving force. This latter point is particularly important since it provides a means to discriminate between rate limiting electron transfer and conformational gating. Nevertheless, chemically modifying redox proteins runs the risk of structural and electrostatic alterations which can be difficult to detect but have profound effects on the redox kinetics. Moreover, the intrinsic protein dynamics can be affected, resulting, in the worst case, in changes in conformational gating which cannot be resolved from rate limiting electron transfer. Given the early stage of development of photo-initiated electron transfer, substantial progress can be expected in the next few years. No doubt new approaches will be developed and existing approaches further refined. Especially important, the theoretical basis for interpreting and understanding electron transfer will continue to evolve.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nonequilibrium dynamics simulations of nitric oxide release: comparative study of nitrophorin and myoglobin

Nitrophorin 4 (NP4) is a heme protein that reversibly binds nitric oxide (NO), with release rates modulated by pH change. High-resolution structures of NP4 revealed that pH changes and NO binding induce a large conformational rearrangement in two loops that serve to protect the heme-bound NO molecule from solvent. We used extended (110 ns) molecular dynamics simulations of NP4 at pH 5 and pH 7, modeled by selective deprotonation of acidic groups. Conformational and dynamic changes were observed, consistent with those found in the crystal. Further, major solvent movement and NO escape were observed at pH 7, while the ligand remained in the heme binding pocket at pH 5. As a control, we also performed molecular dynamics (MD) simulations of sperm whale myoglobin, where NO migration into the interior cavities of the protein was observed, consistent with previous reports. We constructed a kinetic model of ligand escape to quantitatively relate the microscopic rate constants to the observed rates, and tested the predictions against the experimental data. The results suggest that release rates of diatomic molecules from heme proteins can be varied by several orders of magnitude through modest adjustments in geminate rebinding and gating behavior.     

Oxygen activation and electron transfer in flavocytochrome P450 BM3

In flavocytochrome P450 BM3, there is a conserved phenylalanine residue at position 393 (Phe393), close to Cys400, the thiolate ligand to the heme. Substitution of Phe393 by Ala, His, Tyr, and Trp has allowed us to modulate the reduction potential of the heme, while retaining the structural integrity of the enzyme's active site. Substrate binding triggers electron transfer in P450 BM3 by inducing a shift from a low- to high-spin ferric heme and a 140 mV increase in the heme reduction potential. Kinetic analysis of the mutants indicated that the spin-state shift alone accelerates the rate of heme reduction (the rate determining step for overall catalysis) by 200-fold and that the concomitant shift in reduction potential is only responsible for a modest 2-fold rate enhancement. The second step in the P450 catalytic cycle involves binding of dioxygen to the ferrous heme. The stabilities of the oxy-ferrous complexes in the mutant enzymes were also analyzed using stopped-flow kinetics. These were found to be surprisingly stable, decaying to superoxide and ferric heme at rates of 0.01-0.5 s(-)(1). The stability of the oxy-ferrous complexes was greater for mutants with higher reduction potentials, which had lower catalytic turnover rates but faster heme reduction rates. The catalytic rate-determining step of these enzymes can no longer be the initial heme reduction event but is likely to be either reduction of the stabilized oxy-ferrous complex, i.e., the second flavin to heme electron transfer or a subsequent protonation event. Modulating the reduction potential of P450 BM3 appears to tune the two steps in opposite directions; the potential of the wild-type enzyme appears to be optimized to maximize the overall rate of turnover. The dependence of the visible absorption spectrum of the oxy-ferrous complex on the heme reduction potential is also discussed.     

Redox dependent conformational changes in the mixed valence form of the cytochrome c oxidase from p. The reorganization of glutamic acid 278 is coupled to the electron transfer from/to heme a and the binuclear center. denitrificans

In this work we present the separation of FTIR difference signals induced by electron transfer to/from the redox centers of the cytochrome c oxidase from P. denitrificans and compare electrochemically induced FTIR difference spectra with those induced by CO photolysis. FTIR difference spectra of rebinding of CO to the half reduced (mixed valence) form of the cytochrome c oxidase after photolysis reflect the conformational changes induced by the rebinding of CO and by electron transfer reactions from heme a3 to heme a and further on to CUA. During this process, heme a3 (and CUB) are oxidized, whereas heme a and CuA are reduced. By subtracting these difference spectra from an electrochemically induced FTIR difference spectrum, where all four cofactors are reduced, the contributions for heme a3 (and CuB) could be separated. Correspondingly, the spectral contributions of heme a and CuA have been separated. The comparison of these spectra with the spectra calculated for the hemes on the basis of their redox dependent changes previously published in Hellwig et al., (Biochemistry 38, (1999) 1685-1694) show a high degree of similarity, except for additional signals coupled to the reorganization of the binuclear center upon CO rebinding. The separated spectra clearly show that the signals attributed to Glu278, an amino acid discussed to be crucial for proton pumping, is coupled to electron transfer to/from heme a and the binuclear heme a3-CuB center.     

Insight into heme protein redox potential control and functional aspects of six-coordinate ligand-sensing heme proteins from studies of synthetic heme peptides

We describe detailed studies of peptide-sandwiched mesohemes PSMA and PSMW, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSMA with tryptophan (Trp) to give PSMW generates additional energy via Trp side chain-porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His-FeIII ligation to a greater extent than His-FeII ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (Em,8 value). This is intriguing because converting PSMA to PSMW decreased heme solvent exposure, which would normally be expected to stabilize FeII relative to FeIII. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b5. Our studies of the PSMs also revealed that reduction of FeIII to FeII reversed the relative affinities of the first and second His ligands for Fe (K2III > K1III; K2II < K1II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which FeII can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as "dynamic strain", is that an exogenous ligand can compete with one of the His ligands in an FeII-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b5. Exclusive binding of exogenous ligands by the FeII form of PSMA led to positive shifts in its Em,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.     

Mapping the cytochrome C folding landscape

The solution to the riddle of how a protein folds is encoded in the conformational energy landscape for the constituent polypeptide. Employing fluorescence energy transfer kinetics, we have mapped the S.cerevisiae iso-1 cytochrome c landscape by monitoring the distance between a C-terminal fluorophore and the heme during folding. Within 1 ms after denaturant dilution to native conditions, unfolded protein molecules have evolved into two distinct and rapidly equilibrating populations: a collection of collapsed structures with an average fluorophore-heme distance (r) of 27 A and a roughly equal population of extended polypeptides with r > 50 A. Molecules with the native fold appear on a time scale regulated by heme ligation events ( approximately 300 ms, pH 7). The experimentally derived landscape for folding has a narrow central funnel with a flat upper rim on which collapsed and extended polypeptides interchange rapidly in a search for the native structure.     

Quantum design rules for single molecule logic gates

Recent publications have demonstrated how to implement a NOR logic gate with a single molecule using its interaction with two surface atoms as logical inputs [W. Soe et al., ACS Nano, 2011, 5, 1436]. We demonstrate here how this NOR logic gate belongs to the general family of quantum logic gates where the Boolean truth table results from a full control of the quantum trajectory of the electron transfer process through the molecule by very local and classical inputs practiced on the molecule. A new molecule OR gate is proposed for the logical inputs to be also single metal atoms, one per logical input.     

Dynamical regulation of ligand migration by a gate-opening molecular switch in truncated hemoglobin-N from Mycobacterium tuberculosis

Truncated hemoglobin-N is believed to constitute a defense mechanism of Mycobacterium tuberculosis against NO produced by macrophages, which is converted to the harmless nitrate anion. This process is catalyzed very efficiently, as the enzyme activity is limited by ligand diffusion. By using extended molecular dynamics simulations we explore the mechanism that regulates ligand diffusion and, particularly, the role played by residues that assist binding of O2 to the heme group. Our data strongly support the hypothesis that the access of NO to the heme cavity is dynamically regulated by the TyrB10-GlnE11 pair, which acts as a molecular switch that controls opening of the ligand diffusion tunnel. Binding of O2 to the heme group triggers local conformational changes in the TyrB10-GlnE11 pair, which favor opening of the PheE15 gate residue through global changes in the essential motions of the protein skeleton. The complex pattern of conformational changes triggered upon O2 binding is drastically altered in the GlnE11-->Ala and TyrB10-->Phe mutants, which justifies the poor enzymatic activity observed experimentally for the TyrB10-->Phe form. The results support a molecular mechanism evolved to ensure access of NO to the heme cavity in the oxygenated form of the protein, which should warrant survival of the microorganism under stress conditions.