A bis-histidine-ligated unfolded cytochrome c immobilized on anionic SAM shows pseudo-peroxidase activity

Urea-unfolded yeast iso-1-cytochrome c electrostatically adsorbed on a gold electrode coated with an anionic self-assembled monolayer yields a heme-mediated electrocatalytic reduction of H₂O₂ (pseudo-peroxidase activity). Under the same conditions, native cytochrome c is inactive. In the unfolded protein, the Met80 heme iron ligand is replaced by a histidine residue yielding a bis-His-ligated form. H₂O₂ electrocatalysis occurs with an efficient mechanism likely involving direct H₂O₂ interaction with the iron(II) center and formation of a transient ferryl group. Comparison of the catalytic activity of a few urea-unfolded single and double Lys-to-Ala variants shows that the kinetic affinity of H₂O₂ for the heme iron and k_cat of the bis-His-ligated form are strongly affected by the geometry of protein adsorption, controlled by specific surface lysine residues.     

Effect of the electrostatic interaction on the redox reaction of positively charged cytochrome C adsorbed on the negatively charged surfaces of acid-terminated alkanethiol monolayers on a Au(111) electrode

The electrochemical properties of cytochrome c (cyt c) adsorbed on mixed self-assembled monolayers (SAMs) of 2-mercaptoethanesulfonate (MES)/2-mercaptoethanol (MEL) are compared with those on single-component SAMs of MES, MEL, and mercaptopropionic acid (MPA), using cyclic voltammetry and potential-modulated UV-vis reflectance spectroscopy. The rate constant of electron transfer (ET), k(et), of cyt c adsorbed on the SAM of MPA decreases from 1450 +/- 210 s(-1) at pH 7 to 890 +/- 100 s(-1) at pH 9. In contrast, the value of k(et) of cyt c on the SAM of MES is pH-independent at 100 +/- 15 s(-1). Those facts suggest that a large negative charge density on the SAM surface slows down the ET between cyt c and the electrode. The surface charge density of the SAM affects also the amount of electroactive cyt c, Gamma(e), which decreases from 10.0 +/- 1.0 to 5.3 +/- 1.1 pmol cm(-2) with increasing pH from 7 to 9 on the SAM of MPA. Similarly, the k(et) of cyt c adsorbed on the mixed SAMs of MES/MEL sharply decreases from 900 +/- 300 s(-1) to 110 s(-1) as the surface mole fraction of MES increases beyond 0.5, suggesting the presence of a negative surface charge threshold beyond which the rate of ET of cyt c is dramatically lowered. The decrease in the k(et) on the SAMs at high negative charge densities probably results from the confinement of adsorbed cyt c by the strong electrostatic force to an orientation that is not optimal for the ET reaction.     

Photodissociation of heme distal methionine in ferrous cytochrome C revealed by subpicosecond time-resolved resonance Raman spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.     

Reorganization energy of the CuA center in purple azurin: impact of the mixed valence-to-trapped valence state transition

Mixed valence (MV) coordination compounds play important roles in redox reactions in chemistry and biology. Details of the contribution of a mixed valence state to protein electron transfer (ET) reactivity such as reorganization energy, however, have not been experimentally defined. Herein we report measurements of reorganization energies of a binuclear CuA center engineered into Pseudomonas aeruginosa azurin that exhibits a reversible transition between a totally delocalized MV state at pH 8.0 and a trapped valence (TV) state at pH 4.0. The reorganization energy of a His120Ala variant of CuA azurin that displays a TV state at both the above pH values has also been determined. We found that the MV-to-TV state transition increases the reorganization energy by 0.18 eV, providing evidence that the MV state of the CuA center has lower reorganization energy than its TV counterpart. We have also shown that lowering the pH from 8.0 to 4.0 results in a similar (approximately 0.4 eV) decrease in reorganization energy for both blue (type 1) and purple (CuA) azurins, even though the reorganization energies of the two different copper centers are different at a given pH. These results suggest that the MV state plays only a secondary role in modulation of the ET reactivity via the reorganization energy, as compared to that of the driving force.     

Modeling and computations of the intramolecular electron transfer process in the two-heme protein cytochrome c(4)

The di-heme protein Pseudomonas stutzeri cytochrome c(4) (cyt c(4)) has emerged as a useful model for studying long-range protein electron transfer (ET). Recent experimental observations have shown a dramatically different pattern of intramolecular ET between the two heme groups in different local environments. Intramolecular ET in homogeneous solution is too slow (>10 s) to be detected but fast (ms-μs) intramolecular ET in an electrochemical environment has recently been achieved by controlling the molecular orientation of the protein assembled on a gold electrode surface. In this work we have performed computational modeling of the intramolecular ET process by a combination of density functional theory (DFT) and quantum mechanical charge transfer theory to disclose reasons for this difference. We first address the electronic structures of the model heme core with histidine and methionine axial ligands in both low- and high-spin states by structure-optimized DFT. The computations enable estimating the intramolecular reorganization energy of the ET process for different combinations of low- and high-spin heme couples. Environmental reorganization free energies, work terms ("gating") and driving force were determined using dielectric continuum models. We then calculated the electronic transmission coefficient of the intramolecular ET rate using perturbation theory combined with the electronic wave functions determined by the DFT calculations for different heme group orientations and Fe-Fe separations. The reactivity of low- and high-spin heme groups was notably different. The ET rate is exceedingly low for the crystallographic equilibrium orientation but increases by several orders of magnitude for thermally accessible non-equilibrium configurations. Deprotonation of the propionate carboxyl group was also found to enhance the ET rate significantly. The results are discussed in relation to the observed surface immobilization effect and support the notion of conformationally gated ET.     

Probing a complex of cytochrome c and cardiolipin by magnetic circular dichroism spectroscopy: implications for the initial events in apoptosis

Oxidation of cardiolipin (CL) by its complex with cytochrome c (cyt c) plays a crucial role in triggering apoptosis. Through a combination of magnetic circular dichroism spectroscopy and potentiometric titrations, we show that both the ferric and ferrous forms of the heme group of a CL:cyt c complex exist as multiple conformers at a physiologically relevant pH of 7.4. For the ferric state, these conformers are His/Lys- and His/OH(-)-ligated. The ferrous state is predominantly high-spin and, most likely, His/-. Interconversion of the ferric and ferrous conformers is described by a single midpoint potential of -80 ± 9 mV vs SHE. These results suggest that CL oxidation in mitochondria could occur by the reaction of molecular oxygen with the ferrous CL:cyt c complex in addition to the well-described reaction of peroxides with the ferric form.     

Interaction Between Cytochrome c and the Hapten 2,4-Dinitro-fluorobenzene by Electrospray Ionization Mass Spectrometry

Allergic contact dermatitis is a delayed hypersensitivity reaction, which results from skin exposure to low molecular weight chemicals such as haptens. To clarify the pathogenic mechanism, electrospray ionization mass spectrometry (ESI-MS) and hydrogen/deuterium (H/D) exchange, as well as UV spectroscopy, were applied to determine the interaction between the model protein cytochrome c (cyt c) and the hapten 2,4-dinitro-fluorobenzene (DNFB). The ESI-MS results demonstrate that the conformation of cyt c can change from native folded state into partially unfolded state with the increase of DNFB. The equilibrium state H/D exchange followed by ESI-MS further confirms the above results. UV spectroscopy indicates that the strongfield coordination between iron of heme (prosthetic group) and His18 or Met80 of cyt c is not obviously affected by the hapten.     

Prediction of reorganization free energies for biological electron transfer: a comparative study of Ru-modified cytochromes and a 4-helix bundle protein

The acceleration of electron transfer (ET) rates in redox proteins relative to aqueous solutes can be attributed to the protein's ability to reduce the nuclear response or reorganization upon ET, while maintaining sufficiently high electronic coupling. Quantitative predictions of reorganization free energy remain a challenge, both experimentally and computationally. Using density functional calculations and molecular dynamics simulation with an electronically polarizable force field, we report reorganization free energies for intraprotein ET in four heme-containing ET proteins that differ in their protein fold, hydrophilicity, and solvent accessibility of the electron-accepting group. The reorganization free energies for ET from the heme cofactors of cytochrome c and b(5) to solvent exposed Ru-complexes docked to histidine residues at the surface of these proteins fall within a narrow range of 1.2-1.3 eV. Reorganization free energy is significantly lowered in a designed 4-helix bundle protein where both redox active cofactors are protected from the solvent. For all ET reactions investigated, the major components of reorganization are the solvent and the protein, with the solvent contributing close to or more than 50% of the total. In three out of four proteins, the protein reorganization free energy can be viewed as a collective effect including many residues, each of which contributing a small fraction. These results have important implications for the design of artificial electron transport proteins. They suggest that reorganization free energy may in general not be effectively controlled by single point mutations, but to a large extent by the degree of solvent exposure of the ionizable cofactors.     

Protein Unfolding: 1H‐NMR Studies of Paramagnetic Ferricytochrome c‐550 from Horse Heart

Electronic transfer protein cytochrome c‐550 from horse heart is studied in the unfolded state by means of paramagnetic ¹H NMR. The protein contains 104 aminoacid residues and a heme group with low spin Fe^III ion in the oxidized form of protein. The global secondary structure is of the α‐helix type as occurs in the case of very other cytochromes c investigated such as cyt c‐550 from Thiobacillus versutus or cyt c‐551 from Pseudomonas aeruginosa. We have studied the coordination characteristic and electronic properties of heme iron horse heart ferricytochrome c‐550 at increasing denaturing conditions (up to 3.1 M GuHCl and 288‐323 K). The ¹H T₁ values of the signals were measured and some resonance assignments made based on EXSY experiments. The electronic structure of the iron(III) is discussed on the basis of the temperature dependence of the isotropic shifts and relaxation times. These results show that it is produced a change of spin, from low‐spin iron(III) (²T₂, S=1/2) in the folded state to high‐spin iron(III) (⁶A₁, S=5/2) in the unfolded state. It seems to be possible that in the opened structure the ferricyt c‐550 loses one axial ligand (His/‐) appearing the spin transition.     

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.     

Dynamic docking and electron transfer between Zn-myoglobin and cytochrome b(5)

We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4). The rates of photoinitiated ET between the Mb's (1-3) and cyt b(5) have been measured over a range of pH values and ionic strengths. Isothermal titration calorimetry (ITC) and NMR methods have been used to independently investigate the effect of charge neutralization on Mb/b(5) binding. The neutralization of the two heme propionates of ZnMb by formation of the heme diester or, for the first time, the diamide increases the second-order rate constant of the ET reaction between ZnMb and cyt b(5) by as much as several 100-fold, depending on pH and ionic strength, while causing negligible changes in binding affinity. Brownian dynamic (BD) simulations and ET pathway calculations provide insight into the protein docking and ET process. The results support a new "dynamic docking" paradigm for protein-protein reactions in which numerous weakly bound conformations of the docked complex contribute to the binding of cyt b(5) to Mb and Hb, but only a very small subset of these are ET active, and this subset does not include the conformations most favorable for binding; the Mb surface is a large "target" with a small "bullseye" for the cyt b(5) "arrow". This paradigm differs sharply from the more familiar, "simple" docking within a single, or narrow range of conformations, where binding strength and ET reactivity increase in parallel. Likewise, it is distinct from, although complementary to, the well-known picture of conformational control of ET through "gating", or a related picture of "conformational coupling". The new model describes situations in which tight binding does not correlate with efficient ET reactivity, and explains how it is possible to modulate reactivity without changing affinity. Such "decoupling" of reactivity from binding clearly is of physiological relevance for the reduction of met-Mb in muscle and of met-Hb in a red cell, where tight binding of cyt b(5) to the high concentration of ferrous-Mb/Hb would prevent the cytochrome from finding and reducing the oxidized proteins; it likely is of physiological relevance in other situations, as well.     

Photochemical reduction of cytochrome c by a 1,4,5,8-naphthalenediimide radical anion

Steady-state UV irradiation of aqueous solutions containing cytochrome c (cyt c) and N,N'-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (BPNDI), a water-soluble aromatic imide, resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) oxidation state. The reaction kinetics were followed by the increase of the ferrocytochrome c absorbance band at 549 nm. The rate of the photochemical reaction was pH dependent, reaching its maximum values over the pH range 4-7. Addition of electrolyte (NaCl) at pH 5 resulted in a decrease in the reaction rate, as expected for reactions between oppositely charged species. Flash photolysis studies revealed that the actual reductant in the reaction was a photogenerated BPNDI radical anion, which transferred an electron to the cyt c heme iron. The participation of imide radicals in the process was confirmed by the ready reduction of cyt c by BPNDI radicals chemically generated with sodium dithionite.     

吡啶、2-甲基吡啶存在下细胞色素c碱式异构化和配体细胞色素c配合物的电化学研究

用半胱氨酸修饰的金电极研究了吡啶、2 甲基吡啶存在下细胞色素c碱式异构化和配体结合细胞色素c的电化学。在此电极上 ,细胞色素c可发生准可逆的电极反应而吡啶结合细胞色素c和 2 甲基吡啶结合细胞色素c在循环伏安图上只给出还原峰。高浓度 (1.2 7mol·L- 1)的吡啶和 2 甲基吡啶可诱导碱式细胞色素c在中性条件下生成。进一步的研究表明 ,这种诱导作用与配体和细胞色素c的键合无关     

Zinc-porphyrin Solvation in folded and unfolded states of Zn-cytochrome c

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes: the decay in the folded protein (tau(H)2(O) approximately 10 ms) is only modestly affected by isotopically substituted buffers (k(H)2(O)/k(D)2(O) = 1.2), whereas a reduced triplet lifetime (approximately 1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1-0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH(3))(6)(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 x 10(8) M(-1) s(-1) in GdmCl, 6.3 x 10(8) M(-1) s(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 x 10(7) M(-1) s(-1) in GdmCl, 8.6 x 10(6) M(-1) s(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions.     

NO reductase activity of the tetraheme cytochrome C554 of Nitrosomonas europaea

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is believed to function as an electron-transfer protein from hydroxylamine oxidoreductase (HAO). We show here that cyt c(554) also has significant NO reductase activity. The protein contains one high-spin and three low-spin c-type hemes. HAO catalyzed reduction of the cyt c(554), ligand binding, intermolecular electron transfer, and kinetics of NO reduction by cyt c(554) have been investigated. We detect the formation of a NO-bound ferrous heme species in cyt c(554) by EPR and M?ssbauer spectroscopies during the HAO catalyzed oxidation of hydroxylamine, indicating that N-oxide intermediates produced from HAO readily bind to cyt c(554). In the half-reduced state of cyt c(554), we detect a spin interaction between the [FeNO](7) state of heme 2 and the low-spin ferric state of heme 4. We find that ferrous cyt c(554) will reduce NO at a rate greater than 16 s(-1), which is comparable to rates of other known NO reductases. Carbon monoxide or nitrite are shown not to bind to the reduced protein, and previous results indicate the reactions with O(2) are slow and that a variety of ligands will not bind in the oxidized state. Thus, the enzymatic site is highly selective for NO. The NO reductase activity of cyt c(554) may be important during ammonia oxidation in N. europaea at low oxygen concentrations to detoxify NO produced by reduction of nitrite or incomplete oxidation of hydroxylamine.     

Kinetics of histidine deligation from the heme in GuHCl-unfolded Fe(III) cytochrome C studied by a laser-induced pH-jump technique.

We have developed an instrumental setup that uses transient absorption to monitor protein folding/unfolding processes following a laser-induced, ultrafast release of protons from o-nitrobenzaldehyde. The resulting increase in [H(+)], which can be more than 100 microM, is complete within a few nanoseconds. The increase in [H(+)] lowers the pH of the solution from neutrality to approximately 4 at the highest laser pulse energy used. Protein structural rearrangements can be followed by transient absorption, with kinetic monitoring over a broad time range (approximately 10 ns to 500 ms). Using this pH-jump/transient absorption technique, we have examined the dissociation kinetics of non-native axial heme ligands (either histidine His26 or His33) in GuHCl-unfolded Fe(III) cytochrome c (cyt c). Deligation of the non-native ligands following the acidic pH-jump occurs as a biexponential process with different pre-exponential factors. The pre-exponential factors markedly depend on the extent of the pH-jump, as expected from differences in the pK(a) values of His26 and His33. The two lifetimes were found to depend on temperature but were not functions of either the magnitude of the pH-jump or the pre-pulse pH of the solution. The activation energies of the deligation processes support the suggestion that GuHCl-unfolded cyt c structures with non-native histidine axial ligands represent kinetic traps in unfolding.     

Effect of pH on direct electron transfer in the system gold electrode-recombinant horseradish peroxidase.

The effect of pH on the kinetics of the bioelectrocatalytic reduction of H(2)O(2) catalysed by horseradish peroxidase (HRP) has been studied at -50 mV vs. Agmid R:AgCl on HRP-modified Au electrodes placed in a wall-jet flow-through electrochemical cell. Native HRP (nHRP) and a nonglycosylated recombinant form containing a six-histidine tag at the C-terminus, C(His)rHRP, produced by genetic engineering of nonglycosylated recombinant HRP using an E. coli expression system, have been used for adsorptive modification of Au electrodes. A favourable adsorption of C(His)rHRP on pre-oxidized Au from a protein solution at pH 6.0 provided a high and stable current response to H(2)O(2) due to its bioelectrocatalytic reduction based on direct (mediator-less) electron transfer (ET) between Au and the active site of HRP. The heterogeneous ET rate constant, k(s), calculated from experimental data on direct ET, on mediated ET in the presence of catechol as well as from microbalance data, increased more than 30 times when changing from nHRP to C(His)rHRP. For both forms of HRP, the increasing efficiency of bioelectrocatalysis with increasing [H(3)O(+)] was observed. The values of the apparent k(s) between C(His)rHRP and Au changed from a value of 12+/-2 s(-1) in PBS at pH 8.0 to a value of 434+/-62 s(-1) at pH 6.0; a similar k(s)-pH dependence was also observed for nHRP, providing the possibility to consider the reaction mechanism involving the participation of a proton in the rate-determining step of the charge transfer.     

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.     

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.     

Immobilization and electrochemical redox behavior of cytochrome c on fullerene film-modified electrodes

Two different fullerene film-modified electrodes were prepared and used for surface immobilization and electrochemical property investigation of horse heart cytochrome c (cyt c). Both a pristine fullerene film and fullerene-palladium (C(60)-Pd) polymer film-modified platinum, glassy carbon and indium-tin-oxide (ITO) electrodes were used. The immobilized cyt c was characterized by piezoelectric microgravimetry at a quartz crystal microbalance (QCM), UV-visible absorption, and X-ray photoelectron spectroscopy (XPS), as well as cyclic voltammetry (CV) techniques. The UV-visible spectral studies revealed a small blue shift of both the Soret and Q band of the heme moiety of cyt c, immobilized on the C(60)-Pd polymer film-modified ITO electrode, as compared to the bands of cyt c in solution suggesting that molecules of cyt c are densely packed onto the surface of the modified electrode. The CV studies revealed a quasi-reversible electrode behavior of the heme moiety indicating the occurrence of kinetically hindered electron transfer. A good agreement was found between the values of cyt c electrode surface coverage determined by piezoelectric microgravimetry and cyclic voltammetry. For piezoelectric microgravimetry, these values ranged from 0.5 x 10(-10) to 2.5 x 10(-10) mol cm(-2), depending upon the amount of cyt c present in solution and the time allowed for immobilization, which compared with a value of 3.6+/-0.4 x 10(-10) mol cm(-2) determined by CV. The possible mechanisms of cyt c immobilization on the C(60) film and C(60)-Pd film-modified electrodes are also discussed.