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.     

Electrochemistry of unfolded cytochrome c in neutral and acidic urea solutions

The present investigation reports the first experimental measurements of the reorganization energy of unfolded metalloprotein in urea solution. Horse heart cytochrome c (cyt c) has been found to undergo reversible one-electron transfer reactions at pH 2 in the presence of 9 M urea. In contrast, the protein is electrochemically inactive at pH 2 under low-ionic strength conditions in the absence of urea. Urea is shown to induce ligation changes at the heme iron and lead to practically complete loss of the alpha-helical content of the protein. Despite being unfolded, the electron-transfer (ET) kinetics of cyt c on a 2-mercaptoethanol-modified Ag(111) electrode remain unusually fast and diffusion controlled. Acid titration of ferric cyt c in 9 M urea down to pH 2 is accompanied by protonation of one of the axial ligands, water binding to the heme iron (pK(a) = 5.2), and a sudden protein collapse (pH < 4). The formal redox potential of the urea-unfolded six-coordinate His18-Fe(III)-H(2)O/five-coordinate His18-Fe(II) couple at pH 2 is estimated to be -0.083 V vs NHE, about 130 mV more positive than seen for bis-His-ligated urea-denatured cyt c at pH 7. The unusually fast ET kinetics are assigned to low reorganization energy of acid/urea-unfolded cyt c at pH 2 (0.41 +/- 0.01 eV), which is actually lower than that of the native cyt c at pH 7 (0.6 +/- 0.02 eV), but closer to that of native bis-His-ligated cyt b(5) (0.44 +/- 0.02 eV). The roles of electronic coupling and heme-flattening on the rate of heterogeneous ET reactions are discussed.     

Conjugating luminescent CdTe quantum dots with biomolecules

Newly prepared CdTe quantum dots ( QD) bearing shells of water solubility providing capping agents (i.e., thioglycolic acid ( TGA) and 2-(dimethylamino)ethanethiol hydrochloride (DMAET) were subjected to electrostatic assays with several proteins (i.e., cytochrome c (cyt c) and human serum albumin (HSA). In particular, we employed absorption, emission, transient absorption and time-resolved emission spectroscopic means to test their response to light. Only for negatively capped QDs spectroscopic and kinetic evidence were gathered that corroborate the successful bioconjugation of QDs with cyt c to yield QD- cyt c bioconjugates. In fact, photoexcitation of QD-cyt c leads to a fast deactivation of the QD band gap emission and of the QD excited state. Notably, these interactions depend on the size of the QDs. Repulsive forces, on the other hand, are operative between the positively capped QDs and cyt c, hampering any bioconjugation.     

Changes in the heme ligation during folding of a Geobacter sulfurreducens sensor GSU0935

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.     

Kinetics of acid-induced spectral changes in the GFPmut2 chromophore

We have used a nanosecond pH-jump technique, coupled with simultaneous transient absorption and fluorescence emission detection, to characterize the dynamics of the acid-induced spectral changes in the GFPmut2 chromophore. Disappearance of the absorbance at 488 nm and the green fluorescence emission occurs with a thermally activated, double exponential relaxation. To understand the source of the two transients we have introduced mutations in amino acid residues that interact with the chromophore (H148G, T203V, and E222Q). Results indicate that the faster transient is associated with proton binding from the solution, while the second process, smaller in amplitude, is attributed to structural rearrangement of the amino acids surrounding the chromophore. The protonation rate shows a 3-fold increase for the H148G mutant, demonstrating that His148 plays a key role in protecting the chromophore from the solvent. The deprotonation rate for T203V is an order of magnitude smaller, showing that the hydrogen bond with the hydroxyl of Thr203 is important in stabilizing the deprotonated form of the chromophore. A kinetic model suggests that, in addition to protecting the chromophore from the solvent, His148 may act as the primary acceptor for the protons on the way to the chromophore.     

Effects of pH on the kinetic reaction

In most cases, kinetic unfolding reactions of proteins follow a simple one-step mechanism that does not involve any detectable intermediates. One example for a more complicated unfolding reaction is the acid-induced denaturation of holo-myoglobin (hMb). This reaction proceeds through a transient intermediate and can be described by a sequential two-step mechanism (Konermann et al. Biochemistry 1997, 36, 6448-6454). Time-resolved electrospray ionization mass spectrometry (ESI MS) is a new technique for monitoring the kinetics of protein folding and unfolding in solution. Different protein conformations can be distinguished by the different charge state distributions that they generate during ESI. At the same time this technique allows monitoring the loss or binding of noncovalent protein ligands. In this work, time-resolved ESI MS is used to study the dependence of the kinetic unfolding mechanism of hMb on the specific solvent conditions used in the experiment. It is shown that hMb unfolds through a short-lived intermediate only at acidic pH. Under basic conditions no intermediate is observed. These findings are confirmed by the results of optical stopped-flow absorption experiments. This appears to be the first time that a dependence of the kinetic mechanism for protein unfolding on external conditions such as pH has been observed.     

Ruthenium bisbipyridine complexes of horse heart cytochrome c: characterization and comparative intramolecular electron-transfer rates determined by pulse radiolysis and flash photolysis

The reaction of [Ru(bpy)2L(H2O)]2+ (bpy = 2,2'-bipyridine, L = imidazole, water) with reduced horse heart cytochrome c results in coordination of [RuII(bpy)2L] at the His 33 and His 26 sites. Coordination at the His 33 site gave a diastereomeric [RuII(bpy)2L]-His-cyt c(II) mixture favoring the lambda-Ru form regardless of the substituent on the bipyridine ligands, while substitution at the more buried His 26 site gave an isomeric distribution that varies according to the substituent on the bipyridine ligands. The diastereomeric aquoproteins (L = H2O) are distinguished by their redox potentials and their conversion to the corresponding fluorescent imidazole proteins. Intramolecular electron transfer between the reduced ruthenium bipyridine and cyt c(III) in [RuII(bpy.)(bpy)L]-His33-cyt c(III) was determined by reductive pulse radiolysis using the aqueous electron as a reducing agent, kret = (2.0 +/- 0.3) x 10(5) s-1, and kret is independent of the sixth ligand L = H2O, imidazole. In addition, the rate constant for intramolecular electron transfer from cyt c(II) to the ruthenium(III) center in [RuIII(bpy)2L]-His33-cyt c(II) was determined by oxidative pulse radiolysis using azide and carbonate radicals. This rate is very sensitive to the nature of the sixth ligand. When L = H2O, the intramolecular electron-transfer rate for the major diastereomer lambda-cis-[RuIII (bpy)2(H2O)]-His33-cyt c(II) is k = 1.1 x 10(4) s-1 and is independent of pH between 5.6 and 8.3. The minor delta-cis-[RuIII(bpy)2(H2O)]-His33-cyt c(II) isomer has pH-dependent electrochemistry and a lower rate of intramolecular electron transfer. Complete conversion from L = H2O to L = imidazole is slow, requiring more than 7 days in 1 M imidazole. A lower limit (k > 2 x 10(6) s-1) for the intramolecular electron-transfer rate constant in [RuIII(bpy)2(L)]-His33-cyt c(II), L = imidazole, could be obtained by pulse radiolysis in the absence of the slower reacting aquo species. This observation is in agreement with the value of 3 x 10(6) s-1 measured by flash photolysis. Earlier pulse radiolysis experiments primarily measured the aquoligated ruthenium protein, while the flash photolysis experiments measured the imidazole-ligated fraction because it is the only species oxidatively quenched in the photoinduced reactions. Intramolecular electron-transfer reactions for a new series of ruthenium bipyridine complexes, [Ru(dabpy)2L]-His33-cyt c proteins (dabpy = 4,4'-diamino-2,2'-bipyridine) (L = imidazole, pyridine, isonicotinamide and pyrazine), proceed with lower driving force, resulting in slower rate constants amenable to measurement by oxidative pulse radiolysis. The electron-transfer rate constants for this series spanned a wide range of the Marcus log k vs delta G plot.     

The flash-quench technique in protein-DNA electron transfer: reduction of the guanine radical by ferrocytochrome c

Electron transfer from a protein to oxidatively damaged DNA, specifically from ferrocytochrome c to the guanine radical, was examined using the flash-quench technique. Ru(phen)2dppz2+ (dppz = dipyridophenazine) was employed as the photosensitive intercalator, and ferricytochrome c (Fe3+ cyt c), as the oxidative quencher. Using transient absorption and time-resolved luminescence spectroscopies, we examined the electron-transfer reactions following photoexcitation of the ruthenium complex in the presence of poly(dA-dT) or poly(dG-dC). The luminescence-quenching titrations of excited Ru(phen)2dppz2+ by Fe3+ cyt c are nearly identical for the two DNA polymers. However, the spectral characteristics of the long-lived transient produced by the quenching depend strongly upon the DNA. For poly(dA-dT), the transient has a spectrum consistent with formation of a [Ru(phen)2dppz3+, Fe2+ cyt c] intermediate, indicating that the system regenerates itself via electron transfer from the protein to the Ru(III) metallointercalator for this polymer. For poly(dG-dC), however, the transient has the characteristics expected for an intermediate of Fe2+ cyt c and the neutral guanine radical. The characteristics of the transient formed with the GC polymer are consistent with rapid oxidation of guanine by the Ru(III) complex, followed by slow electron transfer from Fe2+ cyt c to the guanine radical. These experiments show that electron holes on DNA can be repaired by protein and demonstrate how the flash-quench technique can be used generally in studying electron transfer from proteins to guanine radicals in duplex DNA.     

Paramagnetic 1H NMR spectrum of nickel(II) pseudoazurin: investigation of the active site structure and the acid and alkaline transitions

The paramagnetic (1)H NMR spectrum of Ni(II) pseudoazurin [(PA)Ni(II)] possesses a number of resonances exhibiting sizable Fermi-contact shifts. These have been assigned to protons associated with the four ligating amino acids, His40, Cys78, His81, and Met86. The shifts experienced by the C(gamma)H protons of the axial Met86 ligand are unprecedented compared to other Ni(II)- and Co(II)-substituted cupredoxins (the C(gamma)(1)H signal is found at 432.5 ppm at 25 degrees C). The large shift of protons of the axial Met86 ligand highlights a strong Ni(II)-S(Met) interaction in (PA)Ni(II). The paramagnetic (1)H NMR spectrum of (PA)Ni(II) is altered by decreasing and increasing the pH value from 8.0. At acidic pH a number of the hyperfine-shifted resonances undergo limited changes in their chemical shift values. This effect is assigned to the surface His6 residue whose protonation results in a structural modification of the active site. Increasing the pH value from 8.0 has a more significant effect on the paramagnetic (1)H NMR spectrum of (PA)Ni(II), and the alkaline transition can now be assigned to two surface lysine residues close to the active site of the protein. The effect of altering pH on the (1)H NMR spectrum of Ni(II) pseudoazurin is smaller than that previously observed in the Cu(II) protein indicating more limited structural rearrangements at the non-native metal site.     

Geminate carbon monoxide rebinding to a c-type haem

A chemically modified form of cytochrome c(cyt. c), termed carboxymethyl cytochrome c(cm cyt. c), possesses a vacant sixth coordination site to the haem iron that is available to bind external ligands. We present data on the rapid flash photolysis of CO from the ferrous haem iron of cm cyt. c and describe the kinetics and spectral transitions that accompany the recombination. This was achieved using 30-femtosecond laser pulses and a white light continuum to monitor spectral transitions. Whereas the photo-dissociation quantum yield is close to 1, the yield of CO escape from the protein (the apparent quantum yield, varphi) relative to myoglobin (varphi=1) is small due to rapid geminate recombination of CO. On ligand photo-dissociation the haem undergoes a spin-state transition from low-spin ferrous CO bound to penta-coordinate high-spin. Subsequently the system reverts to the CO bound form. The data were fitted with a minimum number of exponentials using global analysis. Recombination of CO with the haem iron of cm cyt. c is multiphasic (tau=16 ps, 120 ps and 1 ns), involving three spectrally distinct components. The fraction of haem (0.11) not recombining with CO within 4 ns is similar to the value of varphi(0.12) measured on the same preparation by the "pulse method" (M. Brunori, G. Giacometti, E. Antonini and J. Wyman, Proc. Natl. Acad. Sci. USA, 1973, 70, 3141-3144, ). This implies that no further geminate recombination occurs at t>4 ns. This unusually efficient CO-haem geminate recombination indicates the sterically hindered ("caged") nature of the distal haem pocket in cm cyt. c from which it is difficult for CO to escape. The large geminate phase may be contrasted with the behaviour of myoglobin in which geminate recombination is small. This is in general agreement with the well-documented extensive structural dynamics in myoglobin that allow ligand passage, and a higher structural rigidity in cyt. c imposed by the restraints of minimising reorganisation energy for electron transfer (M. Brunori, D. Bourgeois and D. Vallone, J. Struct. Biol., 2004, 147, 223-234, ). The high pH ferrous form of cm cyt. c is a low-spin species having a lysine bound to the central iron atom of the haem (M. Brunori, M. Wilson and E. Antonini, J. Biol. Chem., 1972, 247, 6076-6081; G. Silkstone, G. Stanway, P. Brzezinski and M. Wilson, Biophys. Chem., 2002, 98, 65-77, ). This high pH (pH approximately 8) form of deoxy cm cyt. c undergoes photo-dissociation of lysine (although the proximal histidine is possible) after photo-excitation. Recombination occurs with a time constant (tau) of approximately 7 ps. This is similar to that observed for the geminate rebinding of the Met80 residue in native ferrous cyt. c(tau approximately 6 ps) following its photo-dissociation (S. Cianetti, M. Negrerie, M. Vos, J.-L. Martin and S. Kruglik, J. Am. Chem. Soc., 2004, 126, 13 932-13 933; W. Wang, X. Ye, A. Demidov, F. Rosca, T. Sjodin, W. Cao, M. Sheeran and P. Champion, J. Phys. Chem., 2000, 104, 10 789-10 801, ).     

Native and unfolded cytochrome c--comparison of dynamics using 2D-IR vibrational echo spectroscopy

Unfolded vs native CO-coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus ( Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs GuHCl concentration produces a sigmoidal curve that overlays the concentration-dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low- and high-concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from subpicosecond to greater than 100 ps through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the approximately 100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy-like state involving hydrophobic collapse around the heme.     

Electroactive multilayer assemblies of bilirubin oxidase and human cytochrome C mutants: insight in formation and kinetic behavior

Here, we report on cytochrome c/bilirubin oxidase multilayer electrodes with different cytochrome c (cyt c) forms including mutant forms of human cyt c, which exhibit different reaction rates with bilirubin oxidase (BOD) in solution. The multilayer formation via the layer-by-layer technique and the kinetic behavior of the mono (only cyt c) and biprotein (cyt c and BOD) multilayer systems are studied by SPR and cyclic voltammetry. For the layer construction, sulfonated polyaniline is used. The only cyt c containing multilayer electrodes show that the quantity of deposited protein and the kinetic behavior depend on the cyt c form incorporated. In the case of the biprotein multilayer with BOD, it is demonstrated that the catalytic signal chain from the electrode via cyt c to BOD and oxygen can be established with all chosen cyt c forms. However, the magnitude of the catalytic current as well as the kinetic behavior differ significantly. We conclude that the different cytochrome c forms affect three parameters, identified here, to be important for the functionality of the multilayer system: the amount of molecules per layer, which can be immobilized on the electrodes, the cyt c self-exchange rate, and the rate constant for the reaction with BOD.     

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     

Autoxidation of ascorbic acid catalyzed by the copper(II) bound to L-histidine oligopeptides, (His)iGly and acetyl-(His)i Gly (i=9, 19, 29). Relationship between catalytic activity and coordination mode

Spectroscopic and kinetic studies on the autoxidation of ascorbic acid catalyzed by copper complexes of histidine oligopeptides, (His)iGly (i=4, 9, 19, 29), and their acetyl derivatives, Ac-(His)iGly (i=9, 19) have been carried out at pH 4.4 and 25 degrees C under dioxygen. The reaction was monitored at 260 nm using a stopped-flow spectrophotometric technique. The reaction fitted the "Michaelis- Menten" mechanism, and ascorbate was oxidized by the "Ping-Pong" mechanism. The Cu(lI) complexed with the oligopeptide (i > or = 9) enhanced the reaction approximately two-fold relative to the aqueous Cu(II). The catalytic activity depends on the molecular weight which is related to the number of histidyl residues and on the coordination mode of the copper-binding site. Results of circular dichroism (CD) experiments revealed the existence of two types of Cu(II). The catalytically active Cu(II), which is accommodated in the imidazole clusters composed of at least six histidyl residues, exhibits d-d transition bands at 520 and 630 nm, and is easily dissociable, enhances the autoxidation; Ac-(His)19Gly is likely to accommodate approximately three active Cu(II) ions. The Cu(II), which is complexed tightly with the terminal H2N-X-Y-His- moiety, where X and Y denote amino acids, inhibits the autoxidation, and exhibits absorption bands at 480 and 550 nm.     

Characterisation of cytochrome c unfolding by nano-electrospray ionization time-of-fight and Fourier transform ion cyclotron resonance mass spectrometry

Protein charge-state distributions (CSDs) in electrospray-ionization mass spectrometry (ESI-MS) represent a sensitive tool to probe different conformational states. We describe here the effect of trifluoroethanol (TFE) on cytochrome c equilibrium unfolding at different pH by nano-ESI-MS. While even low concentrations of TFE destabilize the protein native structure at low pH, a TFE content of 2.5%-5% is found to favor cyt c folding at pH approximately 7. Furthermore, we perform comparison of CSDs obtained by time-of-flight (ToF) and Fourier-transform-ion- cyclotron-resonance (FT-ICR) mass analyzers. To this purpose, we analyze spectra of cyt c in the presence of different kind of denaturants. In particular, experiments with 1-propanol suggest that also by FT-ICR-MS, as previously observed on an ESI-ToF instrument, CSDs do not appear to be controlled by the solvent surface tension as predicted by the Rayleigh-charge model. Moreover, there is general good agreement in conformational effects revealed by the different instruments under several buffer conditions. Nevertheless, the ToF instrument appears to discriminate better between unfolded and partially unfolded forms.     

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.     

Intramolecular vibrational excitation of unfolding reactions in ZnII-substituted and metal-free cytochromes c: activation enthalpies from integrated fluorescence stokes shift and line shape excitation profiles

We have employed continuous-wave fluorescence spectroscopy to observe the light-induced formation of partially unfolded states of Zn(II)-substituted and metal-free (or free-base) cytochrome c (ZnCytc and fbCytc, respectively). In these experiments, the intrinsic porphyrin chromophore provides a vibrational excitation to the protein structure via intramolecular vibrational redistribution of the excess vibronic energy above the first excited singlet state. As the excitation light source is tuned, the fluorescence spectrum of both systems exhibits steplike transitions of the integrated Stokes shift, vibronic structure, and line width that mark apparent activation enthalpy barriers for structural transitions of the protein from the native state to a set of at least three partially unfolded states. The vibronic structure of the ZnCytc spectrum reports the exchange of the Zn(II) ion's native H18 and M80 axial ligands with non-native ligands as the excitation wavenumber is scanned through the three barriers. The metal ion's axial ligands contribute substantially to the stability of ZnCytc; the activation enthalpies for the corresponding transitions in fbCytc are one-third of those in ZnCytc. A comparison of the present results from ZnCytc with those obtained previously with picosecond time-resolved methods [Lampa-Pastirk and Beck, J. Phys. Chem. B 2006, 110, 22971-22974] indicates that the vibrationally excited protein structure propagates along an unfolding pathway from the native state that specifically populates the three states in order of their activation enthalpies. The excitation-wavenumber profile of the fluorescence line width is markedly inconsistent with a Maxwell-Boltzmann distribution over the three states. These results contrast with the general expectation of the protein-folding funnel hypothesis that a distribution of intermediate structures should result from the diffusive propagation of a nonequilibrium protein structure.     

Cytochrome c–dimyristoylphosphatidylglycerol interactions studied by asymmetrical flow field-flow fractionation

Lipid membranes are well recognized ligands that bind peripheral and integral proteins in a specific manner and regulate their function. Cytochrome c (cyt c) is one of the partner peripheral protein that binds to the lipid membranes via electrostatic and hydrophobic interactions. In this study, asymmetrical flow field-flow fractionation (AsFlFFF) was used to compare the interactions of cyt c with the acidic phospholipid 1,2-dimyristoyl-sn-glycero-3-phospho-rac-glycerol (DMPG), oleic acid (OA), and sodium dodecyl sulfate (SDS). The influence of pH and the cyt c–lipid molar mass ratios were evaluated by monitoring the diffusion coefficients and particle diameter distributions obtained for the free and lipid-bound protein. The hydrodynamic particle diameter of cyt c (pI 10) was 4.1 nm at pH 11.4 and around 4.2 nm at pH 7.0 and 8.0. Standard molar mass marker proteins were used for calibration to obtain the molar masses of free cyt c and its complexes with lipids. AsFlFFF revealed the binding of cyt c to DMPG and to OA to be mainly electrostatic. In the absence of electrostatic interactions, minor complex formation occurred, possibly due to the extended lipid anchorage involving the hydrophobic cavity of cyt c and the hydrocarbon chains of DMPG or SDS. The possibility of the formation of the molten globule state of cyt c, induced by the interaction between cyt c and lipids, is discussed.Electronic Supplementary Material Supplementary material is available for this article at     

Enhancing stability and oxidation activity of cytochrome C by immobilization in the nanochannels of mesoporous aluminosilicates

Hydrothermally stable and structrurally ordered mesoporous and microporous aluminosilicates with different pore sizes have been synthesized to immobilize cytochrome c (cyt c): MAS-9 (pore size 90 A), MCM-48-S (27 A), MCM-41-S (25 A), and Y zeolites (7.4 A). The amount of cyt c adsorption could be increased by the introduction of aluminum into the framework of pure silica materials. Among these mesoprous silicas (MPS), MAS-9 showed the highest loading capacity due to its large pore size. However, cyt c immobilized in MAS-9 could undergo facile unfolding during hydrothermal treatments. MCM-41-S and MCM-48-S have the pore sizes that match well the size of cyt c (25 x 25 x 37 A). Hence the adsorbed cyt c in these two medium pore size MPS have the highest hydrothermal stability and overall catalytic activity. On the other hand, the pore size of NaY zeolite is so small that cyt c is mostly adsorbed only on the outer surface and loses its enzymatic activity rapidly. The improved stability and high catalytic activity of cyt c immobilized in MPS are attributed to the electrostatic attraction between the pore surface and cyt c and the confinement provided by nanochannels. We further observed that cyt c immobilized in MPS exists in both high and low spin states, as inferred from the ESR and UV-vis studies. This is different from the native cyt c, which shows primarily the low spin state. The high spin state arises from the replacement of Met-80 ligands of heme Fe (III) by water or silanol group on silica surface, which could open up the heme groove for easy access of oxidants and substrates to iron center and facilitate the catalytic activity. In the catalytic study, MAS-9-cyt c showed the highest specific activity toward the oxidation of polycyclic aromatic hydrocarbons (PAHs), which arises from the fast mass transfer rate of reaction substrate due to its large pore size. For pinacyanol (a hydrophilic substrate), MCM-41-S-cyt c and MCM-48-S-cyt c showed higher specific activity than NaY-cyt c and MAS-9-cyt c. The result indicated that cyt c embedded in the channels of MCM-41-S and MCM-48-S was protected against unfolding and loss of activity. By increasing the concentration of the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) in ESR experiments, we showed that cyt c catalyzes a homolytic cleavage of the O-O bond of hydroperoxide and generates a protein cation radical (g = 2.00). Possible mechanisms for MPS-cyt c catalytic oxidation of hydroperoxides and PAHs are proposed based on the spectroscopic characterizations of the systems.     

Irradiation of the porphyrin causes unfolding of the protein in the protoporphyrin IX/beta-lactoglobulin noncovalent complex

Porphyrins such as protoporphyrin IX (PPIX) are known to occasionally cause conformational changes in proteins for which they are specific ligands. It has also been established that irradiation of porphyrins noncovalently intercalated between bases or bound to one of the grooves can cause conformational effects on DNA. Conversely, there is no evidence reported in the literature of conformational changes caused by noncovalently bound PPIX to globular proteins for which the porphyrin is not a specific ligand. This study shows that the irradiation of the porphyrin in the PPIX/lactoglobulin noncovalent complex indeed causes a local and limited (approximately 7%) unfolding of the protein near the location of Trp19. This event causes the intrinsic fluorescence spectrum of the protein to shift to the red by 2 nm and the average decay lifetime to lengthen by approximately 0.5 ns. The unfolding of lactoglobulin occurs only at pH >7 because of the increased instability of the protein at alkaline pH. The photoinduced unfolding does not depend on the presence of O2 in solution; therefore, it is not mediated by formation of singlet oxygen and is likely the result of electron transfer between the porphyrin and amino acid residues.