Horse Heart Cytochrome Research Articles

Surface Engineering of Gold Nanorods for Cytochrome c Bioconjugation: An Effective Strategy To Preserve the Protein Structure.

The surface of gold nanorods (Au NRs) has been appropriately engineered to achieve a suitable interface for bioconjugation with horse heart cytochrome c (HCc). HCc, an extensively studied and well-characterized protein, represents an ideal model for nanoparticle (NP)–protein conjugation studies because of its small size, high stability, and commercial availability. Here, the native state of the protein has been demonstrated for the first time, by means of Raman spectroscopy, to be retained upon conjugation with the anisotropic Au nanostructures, thus validating the proposed protocol as specifically suited to mostly preserve the plasmonic properties of the NRs and to retain the structure of the protein. The successful creation of such bioconjugates with the retention of the protein structure and function along with the preservation of the NP properties represents a challenging but essential task, as it provides the only way to access functional hybrid systems with potential applications in biotechnology, medicine, and catalysis. In this perspective, the organic capping surrounding the Au NRs plays a key role, as it represents the functional interface for the conjugation step. Cetyltrimethylammonium bromide-coated Au NRs, prepared by using a seed-mediated synthetic route, have been wrapped with polyacrylic acid (PAA) by means of electrostatic interactions following a layer-by-layer approach. The resulting water-dispersible negatively charged AuNRs@PAA NPs have then been electrostatically bound to the positively charged HCc. The bioconjugation procedure has been thoroughly monitored by the combined analysis of UV–vis absorption, resonance Raman and Fourier transform infrared spectroscopies, transmission electron microscopy microscopy, and ζ-potential, which verified the successful conjugation of the protein to the nanorods.

A study on the interaction of horse heart cytochrome c with some conventional and ionic liquid surfactants probed by ultraviolet-visible and fluorescence spectroscopic techniques

The interactions of a protein cytochrome c with some selected conventional and ionic liquid surfactants have been investigated at pH7.4 using ultraviolet-visible and fluorescence spectroscopic techniques. We used four conventional surfactants - cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide (DTAB), sodium N-dodecanoylsarcosinate (SDDS), and N-decanoyl-N-methylglucamine (Mega 10), and a surface active ionic liquid 1-hexadecyl-3-methylimidazolium chloride (C16MeImCl). All the investigated surfactants were found to induce an unfolding of the protein cytochrome c. In presence of CTAB, SDDS and C16MeImCl, the heme iron atom was found to loose methionine from its axial position. Differential binding of the surfactant monomers and their micelles to the protein molecules was inferred. The ionic surfactants were found to be more effective than the nonionic one in unfolding the investigated protein. However, the extent of binding of CTAB/C16MeImCl to cytochrome c reaches a plateau past the critical micellization concentration (cmc) of the surfactant. For each of the cytochrome c-DTAB, cytochrome c-SDDS and cytochrome c-Mega 10 system, although there exists an inflection in the surfactant-binding, saturation point could not be detected. It has been demonstrated from the ultraviolet-visible spectral studies that the oxidation state of iron in cytochrome c does not change when the protein binds with the investigated surfactants.

Porphyrin-Induced Multimerization of Solution-State Proteins

Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome (cyt) PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed formation of multimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY) NMRs for PpcA,.while covalent labeling shielding experiments followed by LC-MS analysis of tryptic digests were used to map ligand binding sites on cyt c, cyt c4 and lysozyme surfaces. All-atom molecular dynamics simulations revealed quick complex formation with binding sites well matching the areas identified in our experimental work. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.

Correction: Reactions of metallodrugs with proteins: selective binding of phosphane-based platinum(ii) dichlorides to horse heart cytochrome c probed by ESI MS coupled to enzymatic cleavage.

Correction for 'Reactions of metallodrugs with proteins: selective binding of phosphane-based platinum(ii) dichlorides to horse heart cytochrome c probed by ESI MS coupled to enzymatic cleavage' by Carolin Mügge et al., Metallomics, 2011, 3, 987-990.

Disentangling of Information About the Structure of Biomolecules Based on the Decomposition and Separation of Two-Dimentional Charge Distributions of Ions

Electrospray ionization mass spectra of biomolecules typically consist of a series of multiply charged ions because of the transfer of protons or other charge carriers between ions of biomolecules and the surrounding liquid or gas. The distribution of intensities of ions retained charge carriers contains information about the spatial structure of biomolecules. A new method is developed for the separation and decomposition of multidimensional charge distributions of ions bearing other charge carriers, such as alkali metal ions, along with protons. The proposed method ensures the estimation of the probability of charge carrier retention by separate functional groups for the selected conformations of biomolecules. The paper describes the application of this method to the analysis of a two-dimensional charge distribution of horse heart cytochrome C, resulting in the revelation of at least two its structural forms under the studied conditions.

Mechanism and Dynamics of Long-Term Stability of Cytochrome c Conferred by Long-Chain Imidazolium Ionic Liquids at Low Concentration

Protein stability has been a concern for researchers for a long time as they are sensitive toward their environment. Mostly proteins during experiments require medium that keep them stable at room temperature (RT). Recent research utilizing ionic liquids (ILs) to stabilize protein has gained much importance. Although a few ILs have been claimed to suit this requirement, reported studies employ IL concentrations that might produce irreversible denaturation and aggregation. This study demonstrates the first report for long-term stabilization of horse heart cytochrome c (h-cyt c) by long-chain imidazolium ILs at far low concentration (1 mM) of IL when stored at RT. Long-chain imidazolium ILs until now were less familiar for their stabilizing nature toward protein. A significant increase in the helical content of h-cyt c (dissolved state) was observed with prolonged structural stability (secondary and tertiary) for about 6 months in aqueous solutions of 1-methyl-3-octyl imidazolium chloride [C8mim][Cl] and 1-...

Cellobiose dehydrogenase hosted in lipidic cubic phase to improve catalytic activity and stability

Lipidic cubic phase systems (LCPs) are excellent carriers for immobilized enzymes due to their biocompatibility and well-defined nanoporous structure. Lipidic cubic phases act as a convenient matrix to incorporate enzymes and hold them in the vicinity of electrode surfaces in their fully active forms. Corynascus thermophilus cellobiose dehydrogenase (CtCDH) was trapped in a monoolein cubic phase, which increased not only its stability, but also its catalytic performance with both enhanced mediated and direct electron transfer with electrodes. For studies of mediated electron transfer, three mediators with different formal potentials (E°′) were employed: horse-heart cytochrome c (cyt c), electron acceptor active with the cytochrome domain of CtCDH, and 2,6-dichlorophenolindophenol (DCPIP) as well as hexaammineruthenium(II) chloride [Ru(NH3)Cl2] both electron acceptors with the dehydrogenase domain. Ru(NH3)Cl2, having the most negative E°′ of −0.138V vs. Ag|AgCl at pH7.5, gave a catalytic current for lactose oxidation of 32.10μAcm−2 in MOPS buffer at pH7.5. The process carried out in the same solution but under direct electron conditions transfer resulted in a catalytic current of 9.22μAcm−2. Electrodes covered with CtCDH in a LCP film retained their catalytic activity after 28days showing a slightly increased current density after 6days.

Porphyrin Induced Multimerization of Solution-State Proteins

Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed formation of multimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear single quantum coherence (HSQC) and total correlation spectroscopy (TOCSY) NMRs for PpcA and cytochrome c, while covalent labeling shielding experiments followed by LC-MS analysis of tryptic digests were used to map ligand binding sites on cyt c4 and lysozyme surfaces. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.

Photoinduced Charge and Energy Transfer in Complexes of C-Type Cytochromes with Water-Soluble Porphyrins

Due to their rich and tunable spectral and redox properties as well as relatively simple synthesis protocols, water-soluble porphyrins are promising candidates as photosensitizers for artificial photosynthesis. Quenching of porphyrin static fluorescence by monoheme horse heart cytochrome (cyt) c is a well-known phenomenon. It is attributed to the fast charge separation (CS), though the CS state has not been observed. Similarly, we found quenching of static fluorescence of porphyrins in the presence of PpcA, a 3-heme c-type cytochrome from the cyt c7 family from Geobacter sulfurreducens. In this report using ultrafast visible transient absorbance spectroscopy we demonstrate that the excited state of protein-bound zinc tetraphenyl sulfonated porphyrin (ZnTPPS) decays with an apparent time constant of about 200 ps in the presence of cyt c and approximately 20 ps in the presence of PpcA. However, the spectral changes are dominated by the features of porphyrin singlet and triplet states. Only a small fraction of photosensitizer excited state energy is retained in the CS state suggesting a competing energy transfer mechanism. Unbound fraction of ZnTPPS shows triplet quenching as a second order process with similar low CS state yields for both cyt c and PpcA. Heteronuclear NMR and all-atom molecular dynamics simulations reveal the likely surface binding sites for ZnTPPS. However, small angle X-ray scattering measurements demonstrate propensity of ZnTPPS to induce formation of protein multimers under experimental conditions typical for our experiments. There results demonstrate an unexpected complexity of ZnTPPS binding interactions and photochemistry with c-type cytochromes and suggest the presence of a competing dominant energy transfer process.

Time resolved calorimetry of photo-induced folding in horse heart cytochrome c at high pH

Here the molar volume and enthalpy changes associated with the early events in the folding of ferrocytochrome c (Cc) at high pH have been examined using time resolved photoacoustic calorimetry (PAC). The data reveal an overall volume change of 1.3 ± 0.3 mL mol−1 and an enthalpy change of 13 ± 7 kcal mol −1 occurring subsequent to photodissociation of the unfolded CO bound Cc species in <∼20 ns. Two additional kinetic phases are observed that are associated with non-native His binding (ΔH and ΔV of 2 ± 4 kcal mol−1 and -0.5 mL mol−1, τ ∼ 2.5 μs ) and Met binding (ΔH and ΔV -0.4 ± 2 kcal mol−1 and -0.1 ± 0.1 mL mol−1, τ∼ 660 ns). Considering only protein conformational changes (excluding volume and enthalpies associated with heme ligation events) the initial conformational event exhibits a ΔH and ΔV of 6 ± 3 kcal mol−1 and -3±0.1 mL mol−1, respectively, that are attributed to a small contraction of the unfolded protein. The corresponding enthalpy associated with both native and non-native ligand binding are found to be −5±4 kcal mol−1 (Fe-Met) and +20 ± 4 kcal mol−1 (Fe-His) with the change in volume for both phases being essential negligible. This would indicate that non-native ligand binding likely occurs from an already collapsed conformation.

Autoxidation of Reduced Horse Heart Cytochrome c Catalyzed by Cardiolipin-Containing Membranes.

Visible circular dichroism, absorption, and fluorescence spectroscopy were used to probe the binding of horse heart ferrocytochrome c to anionic cardiolipin (CL) head groups on the surface of 1,1',2,2'-tetraoleoyl cardiolipin (TOCL)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (20%:80%) liposomes in an aerobic environment. We found that ferrocytochrome c undergoes a conformational transition upon binding that leads to complete oxidation of the protein at intermediate and high CL concentrations. At low lipid concentrations, the protein maintains a structure that is only slightly different from its native one, whereas an ensemble of misligated predominantly hexacoordinated low-spin states become increasingly populated at high lipid concentrations. A minor fraction of conformations with either high- or quantum-mixed-spin states were detected at a CL to protein ratio of 200 (the largest one investigated). The population of the non-native state is less pronounced than that found for cytochrome c-CL interactions initiated with oxidized cytochrome c. Under anaerobic conditions, the protein maintains its reduced state but still undergoes some conformational change upon binding to CL head groups on the liposome surface. Our data suggest that CL-containing liposomes function as catalysts by reducing the activation barrier for a Fe2+ → O2 electron transfer. Adding NaCl to the existing cytochrome-liposome mixtures under aerobic conditions inhibits protein autoxidation of ferrocytochrome c and stabilizes the reduced state of the membrane-bound protein.

Tuning the Selectivity and Reactivity of Metal‐Substituted Polyoxometalates as Artificial Proteases by Varying the Nature of the Embedded Lewis Acid Metal Ion

The hydrolysis of horse heart cytochrome c (cytC) protein by two isostructural Keggin‐type polyoxometalates (POMs), (Me2NH2)10[Ce(α‐PW11O39)2] (Ce1–K2) and (Et2NH2)10[Zr(PW11O39)2] (Zr1–K2), which differ in the nature of the embedded Lewis acid metal ion, has been investigated. In the presence of Ce1–K2, selective hydrolysis of cytC was observed at the Trp60‐Lys61 and Gly78‐Thr79 peptide bonds at pH 7.4 and 37 °C. However, the isostructural Zr1–K2 exhibited a lower reactivity and different selectivity, cleaving cytC at the Asp3‐Val4, Asp51‐Ala52 and Gly78‐Thr79 peptide bonds. Different spectroscopic techniques were used to verify the molecular interactions between cytC and each metal‐substituted Keggin POM to elucidate the role of the Lewis acid metal ion in directing the selectivity of protein hydrolysis.

Refolding of urea denatured cytochrome c: Role of hydrophobic tail of the cationic gemini surfactants

The refolding of urea denatured horse heart cytochrome c (h-cyt-c) under the influence of ester based cationic gemini surfactants [ethane-1, 2-diyl bis(N, N-dimethyl-N-alkylammoniumacetoxy) dichlorides] 16-E2-16, 14-E2-14 and 12-E2-12 (n-E2-n) was performed by using UV–visible, fluorescence and circular dichroism (CD) spectroscopic techniques. We found that n-E2-n geminis promote the formation of molten globule (MG) like state upon addition into the urea denatured h-cyt-c. The comparative study of refolding of denatured h-cyt-c with n-E2-n, cationic gemini surfactant show stabilization of MG-like state influenced by hydrophobic interactions. The formation of MG-like state from the unfolded protein confirms the presence of some regular structures induced by n-E2-n gemini surfactants. Thermodynamic parameters for refolding of h-cyt-c by n-E2-n were also measured and the m-values of all the refolded states of h-cyt-c by n-E2-n show marked difference. The higher m-values correspond to the larger hydrophobic chain length indicates that refolding ability of the n-E2-n depends on the alkyl chain length. The result is related to the stronger hydrophobic forces due to the presence of two head groups and two hydrophobic hydrocarbon tails. This study showed that these cationic gemini surfactants were efficiently utilized in the protein refolding studies.

Chiral Selectivity in Inter-reactant Recognition and Electron Transfer of the Oxidation of Horse Heart Cytochrome c by Trioxalatocobaltate(III).

Outer-sphere electron transfer (ET) between optically active transition-metal complexes and either other transition-metal complexes or metalloproteins is a prototype reaction for kinetic chirality. Chirality as the ratio between bimolecular rate constants of two enantiomers mostly amounts to 1.05-1.2 with either the Λ or Δ form the more reactive, but the origin of chirality in ET parameters such as work terms, electronic transmission coefficient, and nuclear reorganization free energy has not been addressed. We report a study of ET between the Λ-/Δ-[Co(Ox)3](3-) pair (Ox = oxalate) and horse heart cytochrome c (cyt c). This choice is prompted by strong ion-pair formation that enables separation into inter-reactant interaction (chiral "recognition") and ET within the ion pair ("stereoselectivity"). Chiral selectivity was first addressed experimentally. Λ-[Co(Ox)3](3-) was found to be both the more strongly bound and faster reacting enantiomer expressed respectively by the ion-pair formation constant KX and ET rate constant kET(X) (X = Λ and Δ), with KΛ/KΔ and kET(Λ)/kET(Δ) both ≈1.1-1.2. rac-[Co(Ox)3](3-) behavior is intermediate between those of Λ- and Δ-[Co(Ox)3](3-). Chirality was next analyzed by quantum-mechanical ET theory combined with density functional theory and statistical mechanical computations. We also modeled the ion pair K(+)·[Co(Ox)3](3-) in order to address the influence of the solution ionic strength. The complex structure of cyt c meant that this reactant was represented solely by the heme group including the chiral axial ligands L-His and L-Met. Both singlet and triplet hemes as well as hemes with partially deprotonated propionic acid side groups were addressed. The computations showed that the most favorable inter-reactant configuration involved a narrow distance and orientation space very close to the contact distance, substantiating the notion of a reaction complex and the equivalence of the binding constant to a bimolecular reaction volume. The reaction is significantly diabatic even at these short inter-reactant distances, with electronic transmission coefficients κel(X) = 10(-3)-10(-2). The computations demonstrated chirality in both KX and κel(X) but no chirality in the reorganization and reaction free energy (driving force). As a result of subtle features in both KX and κel(X) chirality, the "operational" chirality κET(Λ)KΛ/κET(Δ)KΔ emerges larger than unity (1.1-1.2) from the molecular modeling as in the experimental data.

Activation of Platinum(IV) Prodrugs by Cytochrome c and Characterization of the Protein Binding Sites.

Platinum(IV) complexes generally require reduction to reactive Pt(II) species to exert their chemotherapeutic activity. The process of reductive activation of (15)N-labeled (OC-6-43)-bis(acetato)diamminedichloridoplatinum(IV), in the presence of nicotinamide adenine dinucleotide (NADH) and horse heart cytochrome c (cyt c), was monitored by (1)H,(15)N-HSQC NMR spectroscopy and protein digestion experiments. It has been shown that cyt c plays a catalytic role in the transfer of two reducing equivalents from NADH to Pt(IV) species. Noncovalent interactions between reduced monoaqua cisplatin (cis-[PtCl((15)NH3)2(H2O)](+)) and the protein, in the proximity of the heme cofactor, and also covalent binding of platinum to the protein region around Met65 and Met80 take place.

Cyanide binding to ferrous and ferric microperoxidase-11.

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cytc). MP11 is characterized by a covalently linked solvent-exposed heme group, the heme-Fe atom being axially coordinated by a histidyl residue. Here, the reactions of ferrous and ferric MP11 (MP11-Fe(II) and MP11-Fe(III), respectively) with cyanide have been investigated from the kinetic and thermodynamic viewpoints, at pH 7.0 and 20.0°C. Values of the second-order rate constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 4.5M(-1)s(-1) and 8.9×10(3) M(-1)s(-1), respectively. Values of the first-order rate constant for cyanide dissociation from ligated MP11-Fe(II) and MP11-Fe(III) are 1.8×10(-1)s(-1) and 1.5×10(-3)s(-1), respectively. Values of the dissociation equilibrium constant for cyanide binding to MP11-Fe(II) and MP11-Fe(III) are 3.7×10(-2) and 1.7×10(-7)M, respectively, matching very well with those calculated from kinetic parameters so that no intermediate species seem to be involved in the ligand-binding process. The pH-dependence of cyanide binding to MP11-Fe(III) indicates that CN(-) is the only binding species. Present results have been analyzed in parallel with those of several heme-proteins, suggesting that (1) the ligand accessibility to the metal center and cyanide ionization may modulate the formation of heme-Fe-cyanide complexes, and (2) the general polarity of the heme pocket and/or hydrogen bonding of the heme-bound ligand may affect cyanide exit from the protein matrix. Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c. Penta-coordinated MP11 displays a very high reactivity towards cyanide, whereas the reactivity of hexa-coordinated horse heart cytochrome c is very low.

Effects of Film Morphology and Surface Chemistry on the Direct Electrochemistry of Cytochrome c at Boron-Doped Diamond Electrodes

The effects of film morphology and surface termination on the direct electron transfer of horse heart cytochrome c on boron-doped ultrananocrystalline (B-UNCD) and microcrystalline (B-MCD) diamond thin-film electrodes were investigated. Quasi-reversible, diffusion-controlled cyclic voltammetric responses were observed on oxygen-terminated (atomic O/C ∼0.015), but not hydrogen-terminated (atomic O/C ∼0.02) diamond thin films. The effect of the surface termination was the same for both the nanostructured B-UNCD film with sp2-bonded carbon atoms in the grain boundaries and the well faceted B-MCD film with micron-sized grains and largely devoid of sp2 carbon. Stable cyclic voltammetric i-E curves were recorded with cycling for both oxygen-terminated films indicating the absence of protein denaturation and electrode fouling. The peak currents increased linearly with the square root of the scan rate and the protein concentration; both indicative of a reaction rate limited by semi-infinite linear diffusion of the protein. Similar heterogeneous electron-transfer rate constants were observed for oxygen-terminated B-UNCD (3.48 (±1.25)×10−3cm/s) and B-MCD films (2.38 (±0.72)×10−3cm/s). The results clearly reveal that the oxygen-terminated surface is more active for electron-transfer with this soluble redox protein than is the hydrogen-terminated surface. The film morphology does not influence the diffusion-controlled response of the redox protein.

Multimerization of Solution-State Proteins by Water Soluble Porphyrins

Abstract: Interactions between charged porphyrins and complimentary or similarly charged proteins provide important models systems for studies of electron transfer processes, artificial photosynthesis, and control of protein-protein interactions. Typically, the experimental results are analyzed and discussed assuming that the proteins exist in a monodisperse state. Here, we explored interaction of four solution-state proteins (horse heart cytochrome c, hen egg-white lysozyme, 3-heme c-type cytochrome PpcA from Geobacter sulfurreducens, 2-heme cyt c4 from Pseudomonas stutzeri) with several cationic and anionic water-soluble derivatives of tetraphenylporphyrin. Combined small- and wide-angle X-ray scattering experiments revealed quick formation of mutlimers with a wide range of complex sizes. Thermodynamic interaction parameters and complex binding stoichiometries were established with isothermal calorimetry. Locations of porphyrin binding sites were determined with heteronuclear NMR and chemical modification shielding experiments. The obtained results demonstrate that multimerization of solution-state proteins by large water-soluble ligands appears to be a wide-spread phenomenon controlled by a delicate interplay of electrostatic and hydrophobic forces. Molecular level mapping of the binding sites allows us to build a theory explaining the size of the formed complexes and provides opportunities for targeted design and assembly of multi-subunit protein complexes.

Molecular Simulations of Cytochrome c Adsorption on a Bare Gold Surface: Insights for the Hindrance of Electron Transfer

Experimental studies have shown that the adsorption of horse heart cytochrome c (Cyt-c) on a bare gold surface leads to the hindrance of electron transfer (ET). The underlying mechanism remains controversial. In this work, the adsorption orientation and conformation of Cyt-c on Au(111) surface were investigated by performing Monte Carlo and molecular dynamics simulations. The results show that the most favorable binding mode of Cyt-c to gold agrees with the results characterized by SEIRA spectroscopy. The adsorption is mainly contributed by the strong van der Waals interactions between the surface and residues that have long side chains, which leads to the helix A and Ω1 loop of Cyt-c being in contact with the surface and most of the α-helixes being nearly parallel to the surface. The native structure of Cyt-c is well preserved during the adsorption, and only the flexible Ω1 loop and the N-terminal show a relatively larger mobility. The hindrance of ET is ascribed to the confined rotation of the heme pros...

NO2−-mediated nitrosylation of ferrous microperoxidase-11

Microperoxidase-11 (MP11) is an undecapeptide derived from horse heart cytochrome c (cyt c) and characterized by a covalently-linked solvent-exposed heme group. Here, kinetics of the NO2−-mediated nitrosylation of ferrous MP11 (MP11-Fe(II)) is reported. Data were obtained between pH6.4 and 8.2, at 20.0°C. The NO2−-mediated conversion of MP11-Fe(II) to MP11-Fe(II)-NO requires one proton; accordingly, values of the apparent second-order rate constant (kon) decrease by about two orders of magnitude from (2.9±0.3)×101M−1s−1 to (5.0±0.6)×10−1M−1s−1 upon increasing pH from 6.4 to pH8.2. The slope of the linear fitting of Logkon versus pH is −1.00±0.06. Values of kon for the NO2−-mediated nitrosylation of MP11-Fe(II) are similar to those of penta-coordinated cardiolipin-bound horse heart cyt c, exceeding by about two orders of magnitude those of wild-type horse heart cyt c. Present results highlight the role of heme distal residues in modulating horse heart cyt c reactivity.