Functional biomimetic models for the active site in the respiratory enzyme cytochrome c oxidase

A functional analog of the active site in the respiratory enzyme, cytochrome c oxidase (CcO) reproduces every feature in CcO's active site: a myoglobin-like heme (heme a3), a distal tridentate imidazole copper complex (Cu(B)), a phenol (Tyr244), and a proximal imidazole. When covalently attached to a liquid-crystalline SAM film on an Au electrode, this functional model continuously catalyzes the selective four-electron reduction of dioxygen at physiological potential and pH, under rate-limiting electron flux (as occurs in CcO).     

HYDRATED ELECTRON FORMATION IN NANOSECOND and PICOSECOND LASER FLASH PHOTOLYSIS OF HEMATOPORPHYRIN IN AQUEOUS SOLUTION

Nanosecond (lambda exc = 266, 355 and 532 nm) and picosecond (lambda exc = 355 nm) laser flash photolysis of hematoporphyrin (Hp) was performed in neutral (pH 7.4) and alkaline (pH 12) aqueous solution, as well as in the presence of 0.1% Triton X-100. The dependence of the yield of photoproduced hydrated electrons (e-aq) on laser pulse energy was studied over a wide range of energies (0.2 to greater than 1000 mJ cm-2). The results show that e-aq are predominantly formed in a two-photon process at lambda exc = 266 and 355 nm. One-photon quantum yields are higher at lambda exc = 266 nm than at lambda exc = 355 nm. Both one-photon and two-photon pathways are less efficient at higher Hp concentration, reflecting the influence of Hp self-aggregation. Two-photon e-aq formation is more efficient when 30 ps pulses are used for excitation, as compared to 10 ns pulses. No e-aq could be detected at lambda exc = 532 nm. Nanosecond pulse-induced transient spectra obtained at pH 7.4 are also discussed.     

Parametric investigation of laser-induced fluorescence of solid-state uranyl compounds

The combination of remote/standoff sensing and laser-induced fluorescence (LIF) spectroscopy shows potential for detection of uranyl (UO2(2+)) compounds. Uranyl compounds exhibit characteristic emission in the 450-600 nm (22,200 to 16,700 cm(-1)) spectral region when excited by wavelengths in the ultraviolet or in the short-wavelength portion of the visible spectrum. We report a parametric study of the effects of excitation wavelength [including 532 nm (18,797 cm(-1)), 355 nm (28,169 cm(-1)), and 266 nm (37,594 cm(-1))] and excitation laser power on solid-state uranium compounds. The uranium compounds investigated include uranyl nitrate, uranyl sulfate, uranyl oxalate, uranium dioxide, triuranium octaoxide, uranyl acetate, uranyl formate, zinc uranyl acetate, and uranyl phosphate. We observed the characteristic uranyl fluorescence spectrum from the uranium compounds except for uranium oxide compounds (which do not contain the uranyl moiety) and for uranyl formate, which has a low fluorescence quantum yield. Relative uranyl fluorescence intensity is greatest for 355 nm excitation, and the order of decreasing fluorescence intensity with excitation wavelength (relative intensity/laser output) is 355 nm > 266 nm > 532 nm. For 532 nm excitation, the emission spectrum is produced by two-photon excitation. Uranyl fluorescence intensity increases linearly with increasing laser power, but the rate of fluorescence intensity increase is different for different emission bands.     

Insights into the mechanism of proton transport in cytochrome c oxidase

Cytochrome c oxidase (CcO), known as complex IV of the electron transport chain, plays several important roles in aerobic cellular respiration. Electrons transferred from cytochrome c to CcO's catalytic site reduce molecular oxygen and produce a water molecule. These electron transfers also drive active proton pumping from the matrix (N-side) to intermembrane region (P-side) in mitochondria; the resultant proton gradient activates ATP synthase to produce ATP from ADP. Although the existence of the coupling between the electron transfer and the proton transport (PT) is established experimentally, its mechanism is not yet fully understood at the molecular level. In this work, it is shown why the reduction of heme a is essential for proton pumping. This is demonstrated via novel reactive molecular dynamics (MD) simulations that can describe the Grotthuss shuttling associated with the PT as well as the dynamic delocalization of the excess proton electronic charge defect. Moreover, the "valve" role of the Glu242 residue (bovine CcO notation) and the gate role of d-propionate of heme a(3) (PRDa3) in the explicit PT are explicitly demonstrated for the first time. These results provide conclusive evidence for the CcO proton transporting mechanism inferred from experiments, while deepening the molecular level understanding of the CcO proton switch.     

Efficient photodissociation of O2 from synthetic heme and heme/M (M = Fe, Cu) complexes

Single wavelength excitation (lambdaex = 355 or 532 nm) of low-temperature stabilized (198 K) synthetic heme-dioxygen and heme-dioxygen/M complexes, where M = copper or iron in a non-heme environment, results in the dissociation of dioxygen as indicated by the generation of the ferrous heme (Soret band, 427 nm) and the bleaching of the ferric-superoxide (FeIII(O2-)) 410-nm Soret band in the transient absorption difference spectrum. Dioxygen rebinds to the four heme complexes studied with comparable rate constants ( approximately 6-9 x 105 M-1 s-1). However, the quantum yield for complete dissociation of O2 from our simplest heme-O2 complex (F8)FeIII(O2-) (phi = 0.60) is higher than the other complexes measured (phi = approximately 0.2-0.3) as well as that for oxy-myoglobin (phi = 0.3).     

Multiphoton ionization and Coulomb explosion of C2H5Br clusters: a mass spectrometric and charge density study

Using time‐of‐flight mass spectrometry (TOFMS), laser‐induced photochemistry of ethyl bromide clusters has been investigated at three different wavelengths (viz. 266, 355 and 532 nm) utilizing nanosecond laser pulses of ~5 × 10⁹ W/cm². An interesting finding of the present work is the observation of multiply charged atomic ions of carbon and bromine at 355 and 532 nm, arising from the Coulomb explosion of (C₂H₅Br)_n clusters. At 266 nm, however, the (C₂H₅Br)_n clusters were found to exhibit the usual multiphoton dissociation/ionization behaviour. The TOFMS studies are complemented by measuring the total charge density of the ionized volume at 266, 355 and 532 nm, using the parallel plate method, and the charge densities were found to be ~2 × 10⁹, 6 × 10⁹ and 2 × 10¹¹ charges/cm³, respectively. The significantly higher charge density and the presence of energetic, multiply charged atomic ions at 532 nm are explained by the higher ponderomotive energy of the 532 nm photon, coupled with the Coulomb stability of the residual multiply charged ethyl bromide clusters generated upon laser irradiation, due to their larger effective cluster size at 532 nm than at 355 and 266 nm. Copyright © 2011 John Wiley & Sons, Ltd.     

Spectroscopic characterization and assignment of reduction potentials in the tetraheme cytochrome C554 from Nitrosomonas europaea

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is an essential electron transfer component in the biological oxidation of ammonia. The protein contains one 5-coordinate heme and three bis-His coordinated hemes in a 3D arrangement common to a newly characterized class of multiheme proteins. The ligand binding, electrochemical properties, and heme-heme interactions are investigated with M?ssbauer and X- and Q-band (parallel/perpendicular mode) EPR spectroscopy. The results indicate that the 5-coordinate heme will not bind the common heme ligands, CN(-), F(-), CO, and NO in a wide pH range. Thus, cyt c(554) functions only in electron transfer. Analysis of a series of electrochemically poised and chemically reduced samples allows assignment of reduction potentials for heme 1 through 4 of +47, +47, -147, and -276 mV, respectively. The spectroscopic results indicate that the hemes are weakly exchange-coupled (J approximately -0.5 cm(-)(1)) in two separate pairs and in accordance with the structure: hemes 2/4 (high-spin/low-spin), hemes 1/3 (low-spin/low-spin). There is no evidence of exchange coupling between the pairs. A comparison of the reduction potentials between homologous hemes of cyt c(554) and other members of this new class of multiheme proteins is discussed. Heme 1 has a unique axial N(delta)-His coordination which contributes to a higher potential relative to the homologous hemes of hydroxylamine oxidoreductase (HAO) and the split-Soret cytochrome. Heme 2 is 300 mV more positive than heme 4 of HAO, which is attributed to hydroxide coordination to heme 4 of HAO.     

Transient and stationary spectroscopy of cytochrome c: ultrafast internal conversion controls photoreduction

Photoreduction of cytochrome c (Cyt c) has been reinvestigated using femtosecond-to-nanosecond transient absorption and stationary spectroscopy. Femtosecond spectra of oxidized Cyt c, recorded in the probe range 270-1000 nm, demonstrate similar evolution upon 266 or 403 nm excitation: an ultrafast 0.3 ps internal conversion followed by a 4 ps vibrational cooling. Late transient spectra after 20 ps, from the cold ground-state chromophore, reveal a small but measurable signal from reduced Cyt c. The yield phi for Fe3+-->Fe2+ photoreduction is measured to be phi(403) = 0.016 and phi(266) = 0.08 for 403 and 266 nm excitation. These yields lead to a guess of the barrier E(f)(A) = 55 kJ mol(-1) for thermal ground-state electron transfer (ET). Nanosecond spectra initially show the typical absorption from reduced Cyt c and then exhibit temperature-dependent sub-microsecond decays (0.5 micros at 297 K), corresponding to a barrier E(A)(b) = 33 kJ mol(-1) for the back ET reaction and a reaction energy DeltaE = 22 kJ mol(-1). The nanosecond transients do not decay to zero on a second time scale, demonstrating the stability of some of the reduced Cyt c. The yields calculated from this stable reduced form agree with quasistationary reduction yields. Modest heating of Cyt c leads to its efficient thermal reduction as demonstrated by differential stationary absorption spectroscopy. In summary, our results point to ultrafast internal conversion of oxidized Cyt c upon UV or visible excitation, followed by Fe-porphyrin reduction due to thermal ground-state ET as the prevailing mechanism.     

Planar nitrogen-doped aluminum clusters AlxN- (x=3-5)

The electronic and geometrical structures of three nitrogen-doped aluminum clusters, Al(x)N(-) (x=3-5), are investigated using photoelectron spectroscopy and ab initio calculations. Well-resolved photoelectron spectra have been obtained for the nitrogen-doped aluminum clusters at four photon energies (532, 355, 266, and 193 nm). Global minimum structure searches for Al(x)N(-) (x=3-5) and their corresponding neutrals are performed using several theoretical methods. Vertical electron detachment energies are calculated using three different methods for the lowest energy structures and low-lying isomers are compared with the experimental observations. Planar structures have been established for all the three Al(x)N(-) (x=3-5) anions from the joint experimental and theoretical studies. For Al(5)N(-), a low-lying nonplanar isomer is also found to contribute to the experimental spectra, signifying the onset of two-dimensional to three-dimensional transition in nitrogen-doped aluminum clusters. The chemical bonding in all the planar clusters has been elucidated on the basis of molecular orbital and natural bond analyses.     

The magnetic properties of myoglobin as studied by NMR spectroscopy

Deoxymyoglobin has been investigated by NMR spectroscopy to determine the magnetic anisotropy through pseudocontact shifts and the total magnetic susceptibility through Evans measurements. The magnetic anisotropy values were found to be Deltachi(ax)=-2.03+/-0.08 x 10(-32) m(3) and Deltachi(rh)=-1.02+/-0.09 x 10(-32) m(3). The negative value of the axial susceptibility anisotropy originates from the z tensor axis lying in the heme plane, unlike all other heme systems investigated so far. This magnetic axis is almost exactly orthogonal to the axial histidine plane. The other two axes lie essentially in the histidine plane, the closest to the heme normal being tilted by about 36 degrees from it, towards pyrrole A on the side of the proximal histidine. From the comparison with cytochrome c' it clearly appears that the position of the one axis lying in the heme plane is related to the axial histidine orientation. Irrespective of the directions, the magnetic anisotropy is smaller than that of the analogous reduced cytochrome c' and of the order of that of low-spin iron(III). The magnetic anisotropy of the system permits the measurement of residual dipolar couplings, which, together with pseudocontact shifts, prove that the solution structure is very similar to that in the crystalline state. Magnetic measurements, at variance with previous data, demonstrate that there is an orbital contribution to the magnetic moment, micro(eff)=5.5 micro(B). Finally, from the magnetic anisotropy data, the hyperfine shifts of iron ligands could be separated in pseudocontact and contact components, and hints are provided to understand the spin-delocalisation mechanism in S=2 systems by keeping in mind the delocalisation patterns in low-spin S=1/2 and high-spin S= 5/2 iron(III) systems.     

Electron transfer mechanism and photochemistry of ferrioxalate induced by excitation in the charge transfer band

The photoredox reaction of ferrioxalate after 266/267 nm excitation in the charge transfer band has been studied by means of ultrafast extended X-ray absorption fine structure (EXAFS) analysis, optical transient spectroscopy, and quantum chemistry calculations. The Fe-O bond length changes combined with the transient spectra and kinetics have been measured and in combination with ultrahigh frequency density functional theory (UHF/DFT) calculations are used to determine the photochemical mechanism for the Fe(III) to Fe(II) redox reaction. The present data and the results obtained with 266/267 nm excitations strongly suggest that the primary reaction is the dissociation of the Fe-O bond before intramolecular electron transfer occurs. Low quantum yield electron photodetachment from ferrioxalate has also been observed.     

Orientational control of the physiological reaction of cytochrome c oxidase tethered to a gold electrode

The physiological reaction of a membrane protein is reconstituted on a solid-supported electrode by orientational control via the position of an affinity tag. Recombinant cytochrome c oxidase (CcO) from Rhodobacter sphaeroides is immobilized on a chemically modified gold surface via the affinity of a histidine tag (His-tag) to a nickel chelating nitrilotriacetic acid surface. Control of the orientation is achieved by the adsorption of CcO through the His-tag engineered into the two opposite sites of the membrane protein surface. After reconstitution into a lipid layer, the functionality of this enzyme film electrode is probed by surface-enhanced infrared absorption spectroscopy and cyclic voltammetry. We demonstrate that cytochrome c (Cc) binds and initiates the catalytic reaction of CcO only when the latter is orientated with subunit II facing the bulk aqueous phase while Cc does not interact with the oppositely orientated CcO. We infer from the observed catalytic dioxygen reduction at potentials below 240 mV (vs a normal hydrogen electrode) that reduced Cc mediates electron input into CcO in a way similar to the physiological pathway. The quantitative analysis of the IR spectra indicates the presence of an inactive population of Cc bound to CcO at equal amounts as the redox-active population. This methodological approach demonstrates that the orientation of the membrane protein can be controlled depending on the position of the affinity tag. The approach is considered to be of general applicability as the introduction of affinity tags is routine in current biochemistry.     

Two- and three-wavelength laser multiphoton ionization for highly sensitive detection in solution

Novel two- and three-wavelength laser multiphoton ionization techniques for highly sensitive detection in solution have been established. The photocurrent signal obtained for benzo[a]pyrene by irradiation at 355 nm in n-heptane was effectively enhanced by additional simultaneous irradiation at 532 and/or 1064 nm. The additional irradiation at 532 nm (5 mJ) doubled the signal-to-noise ratio, while that at 1064 nm (30 mJ) increased it 5.5-fold relative to that obtained when only the 355 nm radiation was used. The simultaneous action of 355, 532 (5 mJ) and 1064 (25 mJ) nm radiation further improved the S/N ratio; the detection limit was as low as 1.9 x 10(-10)M. The 532 nm radiation enhanced the photocurrent signal more effectively than did the 1064 nm radiation.     

Horse heart cytochrome c entrapped into the hydrated liquid-crystalline phases of phytantriol: X-ray diffraction and Raman spectroscopic characterization

Small angle X-ray diffraction (SAXD), resonance Raman (RR) spectroscopy with 413 nm excitation, and non-resonance Raman technique with 785 nm excitation were used to probe the influence of entrapped cytochrome c (Cyt c) on the structure of hydrated phytantriol (Phyt) liquid-crystalline phases as well as conformational changes of heme group and secondary structure of the protein. SAXD measurements indicated that incorporation of Cyt c affects both nanostructure dimensions and type of liquid-crystalline phases of hydrated Phyt. The unit cell dimensions decrease with increasing Cyt c concentration for all phases. In addition, protein perturbs the nanostructure of Q(230) and Q(224) liquid-crystalline phases of hydrated Phyt to such an extent that they transform into the Q(229) phase with the Im3m space group. RR data revealed that entrapment of oxidized Cyt c into the Q(230) phase at 1 wt.% content results in near complete reduction of central iron ion of the heme group, while its low-spin state and six-ligand coordination configuration are preserved. Based on the analysis of heme out-of-plane folding vibration near 568 cm(-1) (γ(21)) and ν(48) mode at 633 cm(-1), it was demonstrated that the protein matrix tension on the heme group is relaxed upon incorporation of protein into Q(230) phase. Non-resonant Raman bands of difference spectra showed the preservation of α-helix secondary structure of Cyt c in the liquid-crystalline phase at relatively high (5 wt.%) content. The Cyt c induced spectroscopic changes of Phyt bands were found to be similar as decrease in temperature.     

Photodissociation of p-xylene in polar and nonpolar solutions

The photodissociation of p-xylene at 266 nm in n-heptane and acetonitrile has been studied with use of nanosecond fluorescence and absorption spectroscopy. The p-methylbenzyl radical was identified in n-heptane and acetonitrile by its fluorescence, which was induced by excitation at 308 nm. The p-xylene radical cation was observed in acetonitrile by its absorption. In n-heptane, the decay rate of the S(1) state of p-xylene ((3.2 +/- 0.2) x 10(7) s(-1)) is equal to the growth rate of the p-methylbenzyl radical ((2.7 +/- 0.4) x 10(7) s(-1)), showing that the molecule dissociates via the S(1) state into the radical by C-H bond homolysis (quantum efficiency approximately 5.0 x 10(-3)). In acetonitrile, the formation of the p-xylene radical cation requires two 266 nm photons, and the decay rate of the radical cation ((1.6 +/- 0.2) x 10(6) s(-1)) equals the growth rate of the p-methylbenzyl radical ((2.0 +/- 0.2) x 10(6) s(-1)). This shows that the radical cation dissociates into the radical by deprotonation (quantum efficiency approximately 8.9 x 10(-2)).     

PHOTOREDUCTION OF HORSE HEART CYTOCHROME c

Abstract— During the course of studies on the mechanism of electron transport as catalyzed by cytochrome c , we have found that ferri-horse heart cytochrome c will undergo photoreduction. We have characterized the photoreduction of horse heart cytochrome c in regard to the photochemistry, the nature of the electron donor, and effect of solvent (pH, solvent perturbation, ionic strength). We conclude that ferri-horse heart cytochrome c undergoes photoreduction mediated by a light-induced heme excited state. Further, the protein moiety of cytochrome c serves in part to quench the formation of the excited state, and also to control the interaction of the electron donor and photo-excited cytochrome.     

Wavelength-dependent photochemistry in Cr(CNPh)6: a study of photosubstitution and photoinduced electron transfer using time-resolved spectroscopy

The wavelength dependence of photosubstitution, photoinduced electron transfer, and the time-resolved spectra of Cr(CNPh)6, a compound having low-lying MLCT states, were investigated. Photosubstitution quantum yields increase with increasing excitation energy while photoinduced electron transfer quantum yields decrease with increasing excitation energy. At the lowest excitation energy used (532 nm, or 18,800 cm(-1)), the quantum yields for both electron transfer and photosubstitution reach the same maximum value, 0.29. Picosecond time-resolved absorption spectra at 355 and 532 nm excitation wavelengths show two features: a bleach signal centered at 400 nm and an excited state absorption (ESA) in the 600 nm region. The ESA signal is much weaker for 532 nm excitations than for 355 nm excitations. Following a 355 nm flash, the bleach and ESA decay exponentially with the same lifetime of 23 micros. This implies a simple ligand dissociation followed by recombination. Bleach recovery kinetics after a 532 nm flash are more complicated: two or three exponential components are required to fit the data. Cr(CNPh)6 exhibits two photochemical mechanisms: at high excitation energy, a simple charge neutral dissociation occurs; at low energy, it is proposed that a phenylisocyanide radical anion dissociates, forming a radical pair that is responsible for the observed substitution and electron transfer reactivity, and the complicated nanosecond kinetics. The primary processes for both reactions occur in less than 20 ps.     

Quantum yield of the iodide-iodate chemical actinometer: dependence on wavelength and concentrations

The quantum yield (QY) of the iodide-iodate chemical actinometer (0.6 M KI-0.1 M KIO3) was determined for irradiation between 214 and 330 nm. The photoproduct, triiodide, was determined from the increase in absorbance at 352 nm, which together with a concomitant measurement of the UV fluence enabled the QY to be calculated. The QY at 254 nm was determined to be 0.73 +/- 0.02 when calibration was carried out against a National Institute of Standards and Technology traceable radiometer or photometric device. At wavelengths below 254 nm the QY increased slightly, leveling off at -0.80 +/- 0.05, whereas above 254 nm the QY decreases linearly with wavelength, reaching a value of 0.30 at 284 nm. In addition, the QY was measured at different iodide concentrations. There is a slight decrease in QY going from 0.6 to 0.15 M KI, whereas below 0.15 M KI the QY drops off sharply, decreasing to 0.23 by 0.006 M KI. Calibration of the QY was also done using potassium ferrioxalate actinometry to measure the irradiance. These results showed a 20% reduction in QY between 240 and 280 nm as compared with radiometry. This discrepancy suggests that the QY of the ferrioxalate actinometer in this region of the spectrum needs reexamination.     

Water-soluble polymer-bound biomimetic analogues of cytochrome C oxidase catalyze 4e- reduction of O2 to water

Activity toward catalytic O(2) reduction by polyacrylic acid (PAA) and polyvinylpyridine (PVP) polymers containing functional analogues of the O(2)-reducing site of cytochrome c oxidase (CcO) has been studied in solution and on the electrode surface. Pronounced effects of the porphyrin microenvironment on the selectivity and turnover frequency of the catalysts were observed.     

Effect of electron availability on selectivity of O2 reduction by synthetic monometallic Fe porphyrins

Herein we report that biomimetic analogues of cytochrome c oxidase (CcO) couple reduction of O(2) to oxidation of a single-electron carrier, Ru(NH(3))(6)(2+), under steady-state catalytic turnover. Higher Ru(II) concentrations favor the 4-electron vs 2-electron O(2) reduction pathway. Our data indicate that the capacity of electrode-adsorbed Fe-only porphyrins to catalyze reduction of O(2) to H(2)O is due to high availability of electrons and is eliminated under the biologically relevant slow electron delivery.