Single vesicle observations of the cardiolipin-cytochrome C interaction: induction of membrane morphology changes

We present a novel platform for investigating the composition-specific interactions of proteins (or other biologically relevant molecules) with model membranes composed of compositionally distinct domains. We focus on the interaction between a mitochondrial-specific lipid, cardiolipin (CL), and a peripheral membrane protein, cytochrome c (cyt c). We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and the lipid of interest, CL, preferentially localizes into one of the domains (the liquid disordered (L(d)) phase). The presence of CL-rich and CL-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to CL-rich domains and observe fascinating morphological transitions within these regions of membrane. CL-rich domains start to form small buds and eventually fold up into a collapsed state. We also observe that cyt c can induce a strong attraction between the CL-rich domains of adjacent vesicles as demonstrated by the development of large osculating regions between these domains. Qualitatively similar behavior is observed when other polycationic proteins or polymers of a similar size and net charge are used instead of cyt c. We argue that these striking phenomena can be simply understood by consideration of colloidal forces between the protein and the membrane. We discuss the possible biological implications of our observations in relation to the structure and function of mitochondria.     

Characterization of the Cytochrome c Membrane‐Binding Site Using Cardiolipin‐Containing Bicelles with NMR

Cytochrome (cyt) c transports electrons from Complex III to Complex IV in mitochondria. Cyt c is ordinarily anchored to the mitochondrial membrane through interaction with cardiolipin (CL), however its release into the cytosol initiates apoptosis. The cyt c interaction site with CL‐containing bicelles was characterized by NMR spectroscopy. Chemical shift perturbations in cyt c signals upon interaction with bicelles revealed that a relatively wide region, which includes the A‐site, the CXXCH motif, and the N‐ and C‐terminal helices, and contains multiple Lys residues, interacts cooperatively with CL. The specific cyt c–CL interaction increased with increasing CL molecules in the bicelles. The location of the cyt c interaction site for CL was similar to those for Complex III and Complex IV, thus indicating that cyt c recognizes lipids and partner proteins in a similar way. In addition to elucidating the cyt c membrane‐binding site, these results provide insight into the dynamic aspect of cyt c interactions in mitochondria.     

Specific adsorption of cytochrome C on cardiolipin-glycerophospholipid monolayers and bilayers

In this study, we examined the adsorption of cytochrome c (cyt c) on monolayers and liposomes formed from (i) pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), or cardiolipin (CL) and on (ii) the more thermodynamically stable binary mixtures of POPE/CL (0.8:0.2 mol/mol) and POPC/CL (0.6:0.4 mol/mol). Constant surface pressure experiments showed that the maximum and minimum interactions occurred in the pure CL (anionic phospholipid) and the pure POPE (zwitterion) monolayers, respectively. Observation by atomic force microscopy (AFM) of the images of Langmuir-Blodgett (LB) films extracted at 30 mN m-1 suggests that the different interactions of cyt c with POPE/CL and the POPC/CL monolayers could be due to lateral phase separation occurring in the POPE/CL mixture. The competition between 8-anilino-1-naphthalene sulfonate (ANS) and cyt c for the same binding sites in liposomes that have identical nominal compositions with respect to those of the monolayers was used to obtain binding parameters. In agreement with the monolayer experiments, the most binding was observed in POPE/CL liposomes. All of our observations strongly support the existence of selective adsorption of cyt c on CL, which is modulated differently by different neutral phospholipids (POPE and POPC).     

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.     

Mechanism of superoxide dismutase-like activity of Fe(II) and Fe(III) complexes of tetrakis-N,N,N',N'(2-pyridylmethyl)ethylenediamine

The superoxide dismutase (SOD) activity of iron(II) tetrakis-N,N,N',N'(2-pyridylmethyl)ethylenediamine complex (Fe-TPEN) was reexamined using a pulse radiolysis method. In our previous study (J. Biol. Chem., 264, 9243-9249 (1989)), we reported that this complex has a potent SOD activity in a cyt. c (cytochrome c)-based system (IC50 = 0.8 microM) and protects E. coli cells against paraquat toxicity. The present pulse radiolysis experiment revealed that Fe(II)TPEN reacts stoichiometrically with superoxide to form Fe(III)TPEN with a second-order rate constant of 3.9 x 10(6) M-1 S-1 at pH 7.1, but superoxide did not reduce Fe(III)TPEN to Fe(II)TPEN. The reaction of Fe(III)TPEN and superoxide was biphasic. In the fast reaction, an adduct (Fe(III)TPEN-superoxide complex) was formed at the second-order rate constant of 8.5 x 10(5) M-1 S-1 at pH 7.4. In the slow one, the adduct reacted with another molecule of the adduct, regenerating Fe(III)TPEN. In the cyt. c method with catalase, this Fe(III)TPEN-superoxide complex showed cyt. c oxidation activity, which had led to overestimation of its SOD activity. Based on the titration data, the main species of complex in aqueous media at neutral pH was indicated to be Fe(III)TPEN(OH-). A spectral change after the reduction with hydrated electron indicates that the OH- ion coordinates directly to Fe(III) by displacing one of the pyridine rings. The X-ray analysis of [Fe(II)TPEN]SO4 supported this structure. From the above results we propose a novel reaction mechanism of FeTPEN and superoxide which resembles a proton catalyzed dismuting process, involving Fe(III)TPEN-superoxide complex.     

Electron-transfer reactions through the associated interaction between cytochrome c and self-assembled monolayers of optically active cobalt(III) complexes: molecular recognition ability induced by the chirality of the cobalt(III) units

Self-assembled monolayers (SAMs) of optically active Co(III) complexes ((S)-2/(R)-2) that contain (S)- or (R)-phenylalanine derivatives as a molecular recognition site were constructed on Au electrodes ((S)-2-Au/(R)-2-Au). Molecular recognition characteristics induced by the S and R configurations were investigated by measurements of electron-transfer reactions with horse heart cytochrome c (cyt c). The electrochemical studies indicate that the maximum current of cyt c reduction is obtained when the Au electrode is modified by 2 with a moderate coverage of approximately 4.0 x 10(-11) mol cm(-2). Since the Au electrode is not densely packed with the Co(III) units at this concentration, we conclude that the penetrative association process between cyt c and the Co(III) unit plays an important role in this electron-transfer system. The differences in the electron-transfer rates of (S)-2-Au and (R)-2-Au increase with increasing scan rates, a result indicating that the chiral ligand has an influence on the rate of association of the complexes with cyt c. 3-Au has a mixed monolayer composed of 2 and hexanethiol and exhibits electron-transfer behavior comparable to 2-Au. The difference in the association rates of (S)-3-Au and (R)-3-Au is larger than that between (S)-2-Au and (R)-2-Au, which indicates that the molecular recognition ability of 3-Au has been enhanced by filling the gap between molecules of 2 with hexanethiols. The differences in the oxidation rates of cyt c(II) between (S)-2-Au and (R)-2-Au and between (S)-3-Au and (R)-3-Au were larger than the differences in the rates of the reduction of cyt c(III); this suggests that the size of the heme crevice varies according to the oxidation state of cyt c.     

Characterization of protein-attached conducting polymer monolayer

Cytochrome c (cyt c)-immobilized monolayers and multiple monolayers of a conducting polymer [poly(terthiophene-3-carboxylic acid) polymer (poly-TTCA)] were prepared, where the monolayer of monomer precursor was fabricated with the Langmuir-Blogett technique. Covalent immobilization of cyt c was achieved by the formation of an amide bond between the carboxylic groups of the conducting polymer and amines groups of lysine in cyt c. The monolayer of poly-TTCA and poly-TTCA/cyt c was characterized by cyclic voltammetry, XPS, EQCM, Auger electron spectra (AES), and atomic force microscopy (AFM). The immobilization of cyt c on the polymer layer reveals the direct electron-transfer processes of cyt c. Cyclic voltammetry of the poly-TTCA/cyt c-modified electrode showed a pair of reversible peaks at approximately +212/+201 mV (Epa/Epc) versus Ag/AgCl in a 0.2 M phosphate buffer solution (pH 7.0). The peak separation and the redox peak current of the poly-TTCA/cyt c-modified electrodes were gradually increased by increasing the number of poly-TTCA/cyt c layers on the electrode. The heterogeneous electron-transfer rate constant (ks) of cyt c at the poly-TTCA/cyt c-monolayer-modified electrode was estimated to be 0.874 s(-1). The method provides a novel route for the fabrication of protein (cyt c)-immobilized and/or lipid (palmitoyloleoylphosphatidic acid)-immobilized monolayers and multiple monolayers of a conducting polymer. Cyt c bonded on the conductive polymer layers was applied for bioelectronic devices with unique functionality.     

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     

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.     

Real-time atomic force microscopy reveals cytochrome c-induced alterations in neutral lipid bilayers

The interaction of cytochrome c (cyt c) with supported lipid membranes was investigated on the nanoscale by real-time atomic force microscopy. Cyt c promoted the formation and the expansion of depressed areas in the fluid parts of the bilayer. When the depressions reached the gel domains, they induced the thickening of their edges. According to the step-height differences, cyt c was able to remove neutral lipids in the fluid phase and then to reside on the mica surface. Concerning gel phases, cyt c might insert between the two lipid leaflets, or it might intercalate between the mica and the bilayer.     

Detection of Cytochrome c with Calixarenes Incorporated into Supported Lipid Membranes Via Charge Transient Measurements

The interaction of cytochrome c (cyt c) with the calix[6]arene carboxylic acid derivative (tOct[6]CH2COOH) (CX) incorporated into supported lipid membranes (sBLM) composed of dimyristoylphosphatidylcholine (DMPC) were investigated in the metal/insulator/metal arrangement with the isothermal charge transient spectroscopy (IQTS) in local mode and with voltammetry and measurements of double layer capacitance. Cyt c was detected on hydrated DMPC layers as a distinct IQTS peak with relaxation constant below 100 ms. Incorporation of CX in DMPC increased the sensitivity to cyt c about 50%. Reliable detection limit of cyt c was 30 nM. In sBLMs the capture of cyt c increased the permittivity of both sBLMs, with and without CX, respectively. Nevertheless, cyt c was detected in voltammogram only for DMPC layer contained CX, as a wave at the potential of +0.25 V. This potential is in good agreement with the redox potential of cyt c. Hysteresis in double layer capacitance observed only for the mixed DMPC+CX layer is interpreted by reduction of the charge, which is involved in the ongoing redox reaction.     

Studies on the electrochemical behavior of cytochrome c and its interaction with DNA at a Co/GC ion implantation modified electrode

The electrochemical behavior of cytochrome c (cyt c) and its interaction with DNA at a Co/glassy carbon (GC) ion implantation modified electrode were studied by linear sweep and cyclic voltammetry. In 0.005 mol dm(-3) Tris-0.05 mol dm(-3) NaCl buffer solution (pH = 7.10), a sensitive reduction derivative peak of cyt c was obtained by linear sweep voltammetry. The peak potential was 0.032 V (SCE). The peak current was proportional to the concentration of cyt c. The electrode process was quasi-reversible with adsorption. The electrode reaction rate constant k and the electron transfer coefficient a of cyt c were 4.42 s(-1) and 0.47, respectively. AES and XPS experiments showed that Co was implanted into the surface of the GC electrode (GCE). The implanted Co formed Co-C, which catalyzed the reduction of cyt c. The reaction of DNA with cyt c led to an electrochemically active complex, which resulted in an increase in the reduction current of cyt c. After adding DNA into the solution containing cyt c, the electrode process was still quasi-reversible with adsorption.     

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.     

Effect of mono-CDNP substitution of lysine residues on the redox reaction of cytochrome c electrostatically adsorbed on a mercaptoheptanoic acid modified Au(111) surface

The effect of charge-inverting modification of single surface lysine residue on the electron transfer (ET) reaction of horse heart cytochrome c (cyt c) is examined for 12 different types of mono-4-chloro-2,5-dinitrobenzoic acid substituted cyt c (mCDNPc) adsorbed on a Au(111) electrode modified with a self-assembled monolayer (SAM) of 7-mercapto-heptanoic acid (MHA). A negative shift in the redox potential by 10-35 mV as compared to that of native cyt c and a monolayer coverage in the range of 13-17 pmol cm(-2) are observed for electroactive mCDNPc's. The magnitude of the decrease in the ET rate constant (k(et)) of mCDNPc's compared with that of native cyt c depends on the position of the CDNP substitution. For mCDNPc's in which the modified lysine residue is outside of the interaction domain of cyt c with the SAM, the ratio of the k(et) of mCDNPc to that of native cyt c is correlated to the change in the dipole moment vector of cyt c due to the CDNP modification. This correlation suggests that the dipole moment of cyt c determines its orientation of adsorption on the SAM of MHA and significantly affects the rate of the ET. The CDNP modification of lysine residues at the interaction domain significantly decreases the rate, demonstrating the importance of the local charge environment in determining the rate of ET.     

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.     

Metalloprotein association,self-association,and dynamics governed by hydrophobic interactions: simultaneous occurrence of gated and true electron-transfer reactions between cytochrome f and cytochrome c(6) from Chlamydomonas reinhardtii

Noninvasive reconstitution of the heme in cytochrome c(6) with zinc(II) ions allowed us to study the photoinduced electron-transfer reaction (3)Zncyt c(6) + cyt f(III) --> Zncyt c(6)(+) + cyt f(II) between physiological partners cytochrome c(6) and cytochrome f, both from Chlamydomonas reinhardtii. The reaction kinetics was analyzed in terms of protein docking and electron transfer. In contrast to various protein pairs studied before, both the unimolecular and the bimolecular reactions of this oxidative quenching take place at all ionic strengths from 2.5 through 700 mM. The respective intracomplex rate constants are k(uni) (1.2 +/- 0.1) x 10(4) s(-1) for persistent and k(bi) (9 +/- 4) x 10(2) s(-1) for the transient protein complex. The former reaction seems to be true electron transfer, and the latter seems to be electron transfer gated by a structural rearrangement. Remarkably, these reactions occur simultaneously, and both rate constants are invariant with ionic strength. The association constant K(a) for zinc cytochrome c(6) and cytochrome f(III) remains (5 +/- 3) x 10(5) M(-1) in the ionic strength range from 700 to 10 mM and then rises slightly to (7 +/- 2) x 10(6) M(-1), as ionic strength is lowered to 2.5 mM. Evidently, docking of these proteins from C. reinhardtii is due to hydrophobic interaction, slightly augmented by weak electrostatic attraction. Kinetics, chromatography, and cross-linking consistently show that cytochrome f self-dimerizes at ionic strengths of 200 mM and higher. Cytochrome f(III) quenches triplet state (3)Zncyt c(6), but its dimer does not. Formation of this unreactive dimer is an important step in the mechanism of electron transfer. Not only association between the reacting proteins, but also their self-association, should be considered when analyzing reaction mechanisms.     

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.     

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.     

Thermal unfolding of proteins probed by laser spray mass spectrometry

The stability and conformational changes of cytochrome c (cyt c) at different temperatures and pH have been well examined so far by using various analytical methods. We have found that laser spray mass spectrometry enables much faster and more convenient monitoring of those changes of cyt c compared with other methods. The results correlated well with circular dichroism (CD) experiments under relatively acidic conditions, which destabilize the protein. Laser spray mass spectra of cyt c at various pH were obtained at different levels of laser power. Bimodal charge-state distributions of the protein were observed in laser spray mass spectra, indicating the two-state model of structural change; the lower charges correspond to the folded state, the higher charges to the unfolded state. Based on this result, the presumed denaturation curve of the protein was plotted as a function of laser power, and laser power by which 50% of the protein was assumed to be denatured, E50%, as obtained at each pH. We also examined the melting temperatures, Tm, of cyt c at various values of pH by using CD spectroscopy. The correlation coefficient between E50% and Tm for cyt c was 0.999, demonstrating an excellent correlation. Furthermore, laser spray analysis of ubiquitin, which is found to be more thermally stable than cyt c, gave a higher E50% than cyt c. These results indicate that laser spray mass spectrometry can be an extremely convenient method for probing thermal stabilities and dynamic conformational changes of proteins with subtle structural differences caused by slight changes in pH.     

Controlled morphology and photoreduction characteristics of polyoxometalate(POM)/lipid complexes and the effect of hydrogen bonding at molecular interfaces

Supramolecular complexes consisting of anionic polyoxometalate (POM) and chiral, cationic lipids are newly developed. They give nanofibers, helical ribbons, and nanotapes in organic media depending on the chemical structure of lipid molecules. Lipid ammonium groups exert significant influence on their photoreduction characteristics.