Interheme Electron Transfer Research Articles

Mechanistic Insights on Heme-to-Heme Transmembrane Electron Transfer Within NADPH Oxydases From Atomistic Simulations.

NOX5 is a member of the NADPH oxidase family which is dedicated to the production of reactive oxygen species. The molecular mechanisms governing transmembrane electron transfer (ET) that permits to shuttle electrons over the biological membrane have remained elusive for a long time. Using computer simulations, we report conformational dynamics of NOX5 embedded within a realistic membrane environment. We assess the stability of the protein within the membrane and monitor the existence of cavities that could accommodate dioxygen molecules. We investigate the heme-to-heme electron transfer. We find a reaction free energy of a few tenths of eV (ca. −0.3 eV) and a reorganization free energy of around 1.1 eV (0.8 eV after including electrostatic induction corrections). The former indicates thermodynamically favorable ET, while the latter falls in the expected values for transmembrane inter-heme ET. We estimate the electronic coupling to fall in the range of the μeV. We identify electron tunneling pathways showing that not only the W378 residue is playing a central role, but also F348. Finally, we reveal the existence of two connected O2−binding pockets near the outer heme with fast exchange between the two sites on the nanosecond timescale. We show that when the terminal heme is reduced, O2 binds closer to it, affording a more efficient tunneling pathway than when the terminal heme is oxidized, thereby providing an efficient mechanism to catalyze superoxide production in the final step. Overall, our study reveals some key molecular mechanisms permitting reactive oxygen species production by NOX5 and paves the road for further investigation of ET processes in the wide family of NADPH oxidases by computer simulations.

Transmembrane Hemoprotein Optical Reporters (THORs) for Membrane Potential Sensing

Engineering probes that can image changes in membrane potential with high spatial and temporal resolution is essential for enhancing our understanding of the mechanisms that underlie neural activity. While organic voltage sensitive dyes report voltage signaling events with high sensitivity and temporal resolution, they lack the ability to be targeted genetically to specific cell types. Genetically encoded voltage indicators (GEVIs) can overcome this targeting problem; however, GEVIs tend to be both slower and dimmer when compared to voltage sensitive dyes. Here, we present our progress on the development and characterization of a novel family of GEVIs to improve the properties of current GEVIs. Our GEVIs are called Transmembrane Hemoprotein Optical Reporters (THORs), which consist of a transmembrane 4-α-helical protein and a fluorescent protein. The core of THORs binds heme cofactors, whose oxidation states are affected by the transmembrane electric field. The speed of inter-heme electron transfer is strongly dependent on the distance between cofactors. Based on the Moser-Dutton Ruler of electron transfer, we can achieve electron transfer rates on the µs timescales between heme cofactors. A fluorescent protein reports the change in redox state of one of the hemes via FRET. Water-soluble prototypes of THORs expressed in E. coli revealed an 18% change in fluorescence upon heme reduction for mOrange2 and a 59% change for Sirius. Two different lengths of THORs were designed: one whose length barely exceeds the thickness of a membrane and a second one which extends into the cytoplasm. THORs have been expressed in HEK293t cells and rat hippocampal neurons. In rat hippocampal neurons, a depolarization of the membrane via potassium gradient decreased the fluorescence of THORs, as expected. Currently, we are using whole cell patch clamp techniques to determine the temporal response of THORs.

Regulation of cytochrome c oxidase activity by modulation of the catalytic site

The respiratory supercomplex factor 1 (Rcf 1) in Saccharomyces cerevisiae binds to intact cytochrome c oxidase (CytcO) and has also been suggested to be an assembly factor of the enzyme. Here, we isolated CytcO from rcf1Δ mitochondria using affinity chromatography and investigated reduction, inter-heme electron transfer and ligand binding to heme a3. The data show that removal of Rcf1 yields two CytcO sub-populations. One of these sub-populations exhibits the same functional behavior as CytcO isolated from the wild-type strain, which indicates that intact CytcO is assembled also without Rcf1. In the other sub-population, which was shown previously to display decreased activity and accelerated ligand-binding kinetics, the midpoint potential of the catalytic site was lowered. The lower midpoint potential allowed us to selectively reduce one of the two sub-populations of the rcf1Δ CytcO, which made it possible to investigate the functional behavior of the two CytcO forms separately. We speculate that these functional alterations reflect a mechanism that regulates O2 binding and trapping in CytcO, thereby altering energy conservation by the enzyme.

Bidirectional Photoinduced Electron Transfer in Ruthenium(II)-Tris-bipyridyl-Modified PpcA, a Multi-heme c-Type Cytochrome from Geobacter sulfurreducens.

PpcA, a tri-heme cytochrome c7 from Geobacter sulfurreducens, was investigated as a model for photosensitizer-initiated electron transfer within a multi-heme "molecular wire" protein architecture. Escherichia coli expression of PpcA was found to be tolerant of cysteine site-directed mutagenesis, demonstrated by the successful expression of natively folded proteins bearing cysteine mutations at a series of sites selected to vary characteristically with respect to the three -CXXCH- heme binding domains. The introduced cysteines readily reacted with Ru(II)-(2,2'-bpy)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form covalently linked constructs that support both photo-oxidative and photo-reductive quenching of the photosensitizer excited state, depending upon the initial heme redox state. Excited-state electron-transfer times were found to vary from 6 × 10(-12) to 4 × 10(-8) s, correlated with the distance and pathways for electron transfer. The fastest rate is more than 10(3)-fold faster than previously reported for photosensitizer-redox protein constructs using amino acid residue linking. Clear evidence for inter-heme electron transfer within the multi-heme protein is not detected within the lifetimes of the charge-separated states. These results demonstrate an opportunity to develop multi-heme c-cytochromes for investigation of electron transfer in protein "molecular wires" and to serve as frameworks for metalloprotein designs that support multiple-electron-transfer redox chemistry.

Approach to Interfacial and Intramolecular Electron Transfer of the Diheme Protein Cytochrome c4 Assembled on Au(111) Surfaces

Intramolecular electron transfer (ET) between metal centers is a core feature of large protein complexes in photosynthesis, respiration, and redox enzyme catalysis. The number of microscopic redox potentials and ET rate constants is, however, prohibitive for experimental cooperative ET mapping, but two-center proteins are simple enough to offer complete communication networks. At the same time, multicenter redox proteins operate in membrane environments where conformational dynamics may lead to gated ET features different from conditions in homogeneous solution. The bacterial respiratory diheme protein Pseudomonas stutzeri cytochrome c(4) has been a target for intramolecular, interheme ET. We report here voltammetric and in situ scanning tunneling microscopy (STM) data for P. stutzeri cyt c(4) at single-crystal, atomically planar Au(111)-electrode surfaces modified by variable-length omega-mercapto-alkanoic carboxylic acids. As evidenced by in situ STM, the strongly dipolar protein is immobilized in a close to vertical orientation at this surface with the positively charged high-potential heme domain adjacent to the electrode. This orientation gives asymmetric voltammograms with two one-ET peaks in the cathodic direction and a single two-ET peak in the anodic direction. Intramolecular, interheme ET with high, 8,000-30,000 s(-1), rate constants is notably an essential part of this mechanism. The high rate constants are in striking contrast to ET reactions of P. stutzeri cyt c(4) with small reaction partners in homogeneous solution for which kinetic analysis clearly testifies to electrostatic cooperative effects but no intramolecular, interheme ET higher than 0.1-10 s(-1). This difference suggests a strong gating feature of the process. On the basis of the three-dimensional structure of P. stutzeri cyt c(4), gating is understandable due to the through-space, hydrogen-bonded electronic contact between the heme propionates which is highly sensitive to environmental configurational fluctuations.

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c(4) by [Co(bipy)(3)](2+/3+) (bipy=2,2'-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both "cooperative"intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c(4) is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c(4) for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less "ballistic" modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c(4) function.

Quantum Mechanical Investigations of Heme Structure and Vibrational Spectra: Effects of Conformation, Oxidation State, and Electric Field

Three conformers of a complex consisting of a Fe-porphyrin ring with axial imidazole groups FeP(Im)(2) that differed by orientation of axial imidazole groups were studied with quantum mechanical calculations in both reduced (neutral) and oxidized (cationic) states. In the reduced state, all three conformers correspond to local minima. On the other hand, in the oxidized state the conformer with the imidazole groups perpendicular to one another does not have a local minimum, which suggests that there could be changes in the structure of heme groups depending on oxidation state. The relative energy differences between conformers are small as well the differences of orbital energies. However, the populations of Fe 3d orbitals, and hence charge distributions, are predicted to change significantly between conformations. The orientation of heme groups can affect the kinetics of interheme electron transfer, so this electronic population redistribution will affect electron transfer kinetics. A comparison of calculated Raman spectra with measured surface-enhanced Raman spectra (Biju et al. Langmuir 2007, 23, 1333) shows excellent agreement for frequencies, but correlations of Raman intensities are less satisfactory. Possible explanations of the observed discrepancies could arise due to a problem in assignments of vibration bands in the experiment, a dependence of Raman spectra on heme complex on oxidation state, long-range protein structure, or membrane electric field are discussed. In particular, it was shown that the oxidation state does not dramatically alter the calculated Raman spectrum. However, the cell membrane electric field can significantly modify Raman spectra. An interpretation of the experimental oxidized cytochrome surface-enhanced Raman spectra is discussed.

Effect of isotope substitution and controlled dehydration on the photoinduced electron transport reactions of quinone acceptors and multiheme cytochrome C in bacterial photosynthetic reaction center.

Isotope substitution of H2O by 2H2O causes an increase in the rate of dark recombination between photooxidized bacteriochlorophyll (P+) and reduced primary quinone acceptor in Rhodobacter sphaeroides reaction centers (RC) at room temperature. The isotopic effect declines upon decreasing the temperature. Dehydration of RC complexes of Ectothiorhodospira shaposhnikovii chromatophores containing multiheme cytochrome c causes a decrease in the efficiency of transfer of a photomobilized electron between the primary and secondary quinone acceptors and from cytochrome to P+. In the case of H2O medium these effects are observed at a lower hydration than in 2H2O-containing medium. In the E. shaposhnikovii chromatophores subjected to dehydration in H2O, the rate of electron transfer from the nearest high-potential cytochrome heme to P+ is virtually independent of hydration within the P/P0 range from 0.1 to 0.5. In samples hydrated in 2H2O this rate is approximately 1.5 times lower than in H2O. However, the isotopic effect of this reaction disappears upon dehydration. The intramolecular electron transfer between two high-potential hemes of cytochrome c in samples with 2H2O is inhibited within this range of P/P0, whereas in RC samples with H2O there is a trend toward gradual inhibition of the interheme electron transfer with dehydration. The experimental results are discussed in terms of the effects of isotope substitution and dehydration on relaxation processes and charge state of RC on implementation of the reactive states of RC providing electron transfer control.

Resonance Raman and surface-enhanced resonance Raman studies of polymer-modified electrodes which mimic heme enzymes

Iron-5,10,15,20-tetraphenylporphyrin (FeTPP) has been incorporated into films of a coordinating hydrogel polymer support medium, poly(γ-ethyl-L-glutamate) (PEG) functionalised with imidazole pendant arms (PEG-Im), and studied insitu on silver electrodes using a combination of both resonance Raman (RR) and surface-enhanced resonance Raman (SERR) spectroscopy. The SERR spectra give information on the portion of the film close to the electrode surface while RR spectra probe the '‘bulk’' of the film. At open-circuit potentials the RR spectra are characteristic of the expected low-spin FeIII(TPP)(PEG-Im)2 complex formed by axial ligation but the SERR spectra show that, at the electrode surface, the complex is composed primarily of FeIII(TPP)(PEG-Im). The reasons for the difference have been investigated by systematic RR and SERR studies of both PEG-Im and a more inert polymer support based on simple PEG, which does not carry any potentially ligating imidazole pendant arms. On application of a reducing potential (-400 mV vs. SSCE) only partial reduction is observed at the surface of the PEG-Im films. However, RR spectra of the reduced films show complete and reversible conversion to the expected FeII(TPP)(PEG-Im)2 complexes so that the low electrochemical activity near the surface does not prevent efficient electron transport from the electrode surface right through the thickness of the doped polymer layer. There are striking similarities between the properties of this model system, which contains multiple randomly oriented iron porphyrins which are bis-axially coordinated by imidazoles in a solvent accessible poly(amino acid) matrix, and those of cytochrome c3, which is a tetraheme protein of low molecular weight. In cyt c3 the FeIII hemes, which are bis-coordinated by histidine residues, lie close to each other and again show efficient inter-heme electron transfer. The structure of the synthetic PEG-Im film model appears to be sufficiently close in structure to the enzyme that it also reproduces the main features of its behaviour.

Pulse radiolysis studies on cytochrome cd1 nitrite reductase from Thiosphaera pantotropha: evidence for a fast intramolecular electron transfer from c-heme to d1-heme.

Electron transfer within cytochrome cd1 from Thiosphaera pantotropha was investigated by the technique of pulse radiolysis. The reduction of the heme centers in this nitrite reductase occurred in two phases as judged from kinetic difference spectra. In the faster phase, radiolytically generated N-methylnicotinamide (NMA) radicals selectively reduced the c-heme of the enzyme. From the absorbance increase at 420 nm, a characteristic of formation of the ferrousc-heme, the second-order rate constant for this electron transfer process was estimated to be 3.8 x 10(9) M-1 s-1 at pH 7.0. In the slower phase, a decrease of absorption around 420 and 550 nm, corresponding to a reoxidation of the c-heme, was accompanied by an increase of absorption around 460 and 640 nm, characteristic of formation of the reduced d1-heme. This indicated that an intramolecular electron transfer from the c-heme to the d1-heme occurred. The first-order rate constant of this process was calculated to be 1.4 x 10(3) s-1 at pH 7.0 and was independent of the enzyme concentration. In the presence of nitrite the interheme electron transfer rate was not affected, but on a time scale of seconds a new species associated with the d1-heme, having an absorption maximum at 640 nm, was detected and is proposed to reflect ligand binding to this heme. These results suggest the role of the c-heme as the electron acceptor site in cytochrome cd1 and in mediating the electron transfer to the catalytic site of the enzyme. Moreover, the fast interheme electron transfer rate argues against this process being the rate determining step in catalysis.

Oxygen Binding and Activation: Early Steps in the Reaction of Oxygen with Cytochrome c Oxidase

We have studied the flow-flash reaction of fully reduced cytochrome c oxidase with a high concentration of oxygen (1 mM), recording the first 200 microseconds of the reaction at a number of wavelengths between 400 and 455 nm. This approach has allowed us to observe kinetic phases with time constants of 8 and 32 microseconds and to separate their spectra. The spectrum of the first phase is comparable to that of oxygen binding to myoglobin, while the spectrum of the second phase appears to contain a contribution from a peroxy intermediate. The results are discussed in the context of a model in which the 8-microseconds phase reflects the establishment of an equilibrium of oxygen-bound states, while the 32-microseconds phase arises when this system is trapped as a peroxy intermediate, by inter-heme electron transfer.