Heme Group Of Cytochrome Research Articles

Probing the orientation of electrostatically immobilized cytochrome C by time of flight secondary ion mass spectrometry and sum frequency generation spectroscopy

By taking advantage of the electron pathway through the heme group in cytochrome c (CytoC) electrochemists have built sensors based upon CytoC immobilized onto metal electrodes. Previous studies have shown that the electron transfer rate through the protein is a function of the position of this heme group with respect to the electrode surface. In this study a detailed examination of CytoC orientation when electrostatically immobilized onto both amine (NH3+) and carboxyl (COO-) functionalized gold is presented. Protein coverage, on both surfaces, was monitored by the change in the atomic % N, as determined by x-ray photoelectron spectroscopy. Spectral features within the in situ sum frequency generation vibrational spectra, acquired for the protein interacting with positively and negatively charged surfaces, indicates that these electrostatic interactions do induce the protein into a well ordered film. Time of flight secondary ion mass spectrometry data demonstrated a clear separation between the two samples based on the intensity differences of secondary ions stemming from amino acids located asymmetrically within CytoC (cysteine: C2H6NS+; glutamic acid: C4H6NO+ and C4H8NO2+; leucine: C5H12N+). For a more quantitative examination of orientation, we developed a ratio comparing the sum of the intensities of secondary-ions stemming from the amino acid residues at either end of the protein. The 50 % increase in this ratio, observed between the protein covered NH3+ and COO- substrates, indicates opposite orientations of the CytoC on the two different surfaces.

Capillary electrophoresis–mass spectrometry of intact basic proteins using Polybrene–dextran sulfate–Polybrene-coated capillaries: System optimization and performance

A capillary electrophoresis–mass spectrometry (CE–MS) method using sheath liquid electrospray ionization interfacing was studied and optimized for the analysis of intact basic proteins. To prevent protein adsorption, capillaries with a noncovalent positively charged coating were utilized. Capillaries were coated by subsequent rinsing with solutions of Polybrene, dextran sulfate and Polybrene. The coating proved to be fully compatible with MS detection, causing no background signals and ionization suppression. The composition of the sheath liquid and BGE was optimized using the model proteins α-chymotrypsinogen A, ribonuclease A, lysozyme and cytochrome c. A sheath liquid of isopropanol–water–acetic acid (75:25:0.1, v/v/v) at 2 μL min −1 resulted in optimal signal intensities for most proteins, but caused dissociation of the heme group of cytochrome c. Optimum protein responses were obtained with a BGE of 50 mM acetic acid (pH 3.0), which allowed a baseline separation of the test protein mixture. Several minor impurities present in the mixture could be detected and provisionally identified using accurate mass and a protein modification database. The selectivity of the CE–MS system was investigated by the analysis of acetylated lysozyme. Eight highly related species, identified as non-acetylated lysozyme and lysozyme acetylated in various degrees, could be distinguished. The CE–MS system showed good reproducibility yielding interday (three weeks period) RSDs for migration time and peak area within 2% and 10%, respectively. With the CE–MS system, determination coefficients ( R 2) for protein concentration and peak area were higher than 0.996, whereas detection limits were between 11 and 19 nM.

X-ray absorption spectroscopy of biomimetic dye molecules for solar cells

Dye-sensitized solar cells are potentially inexpensive alternatives to traditional semiconductor solar cells. In order to optimize dyes for solar cells we systematically investigate the electronic structure of a variety of porphyrins and phthalocyanines. As a biological model system we use the heme group in cytochrome c which plays a role in biological charge transfer processes. X-ray absorption spectroscopy of the N 1s and C 1s edges reveals the unoccupied molecular orbitals and the orientation of the molecules in thin films. The transition metal 2p edges reflect the oxidation state of the central metal atom, its spin state, and the ligand field of the surrounding N atoms. The latter allows tuning of the energy position of the lowest unoccupied orbital by several tenths of an eV by tailoring the molecules and their deposition. Fe and Mn containing phthalocyanines oxidize easily from +2 to +3 in air and require vacuum deposition for obtaining a reproducible oxidation state. Chlorinated porphyrins, on the other hand, are reduced from +3 to +2 during vacuum deposition at elevated temperatures. These findings stress the importance of controlled thin film deposition for obtaining photovoltaic devices with an optimum match between the energy levels of the dye and those of the donor and acceptor electrodes, together with a molecular orientation for optimal overlap between the pi orbitals in the direction of the carrier transport.

Cytochrome c induces lipid demixing in weakly charged phosphatidylcholine/phosphatidylglycerol model membranes as evidenced by resonance energy transfer

Resonance energy transfer (RET) between anthrylvinyl-labeled phosphatidylcholine (AV-PC) or phosphatidylglycerol (AV-PG) as donors and the heme groups of cytochrome c (cyt c) as acceptors was examined in PC/PG model membranes containing 10, 20 or 40 mol% PG with an emphasis on evaluating lipid demixing caused by this protein. The differences between AV-PC and AV-PG RET profiles observed at PG content 10 mol% were attributed to cyt c ability to produce segregation of acidic lipids into lateral domains. The radius of lipid domains recovered using Monte-Carlo simulation approach was found not to exceed 4 nm pointing to the local character of cyt c-induced lipid demixing. Increase of the membrane PG content to 20 or 40 mol% resulted in domain dissipation as evidenced by the absence of any RET enhancement while recruiting AV-PG instead of AV-PC.

Dynamic consequences of mutating the typical HPGG motif of apocytochrome b5 revealed by computer simulation

Apocytochrome b 5 with a typical heme-binding motif of HPGG, and its variants with mutated motifs, GPGG, GPGH, HVGG, and HPGP, have been subjected to molecular dynamics simulation. Comparison of the dynamic consequences has revealed the crucial role of HPGG in assembling the heme group of cytochrome b 5 and in modulating protein structure, property and function.

The binding of cytochrome c to neuroglobin: A docking and surface plasmon resonance study

It has recently been proposed that the role of neuroglobin in the protection of neurons from ischaemia induced cell death requires the formation of a transient complex with cytochrome c. No such complex has yet been isolated. Here, we present the results of soft docking calculations, which indicate one major binding site for cytochrome c to neuroglobin. The results yield a plausible structure for the most likely complex structure in which the hemes of each protein are in close contact. NMR analysis identifies the formation of a weak complex in which the heme group of cytochrome c is involved. surface plasmon resonance studies provide a value of 45 μM for the equilibrium constant for cytochrome c binding to neuroglobin, which increases significantly as the ionic strength of the solution increases. The temperature dependence of the binding constant indicates that the complex formation is associated with a small unfavourable enthalpy change (1.9 kcal mol −1) and a moderately large, favourable entropy change (14.8 cal mol −1 deg −1). The sensitivity of the binding constant to the presence of salt suggests that the complex formation involves electrostatic interactions.

Structure of the Intermolecular Complex between Plastocyanin and Cytochrome f from Spinach

In oxygenic photosynthesis, plastocyanin shuttles electrons between the membrane-bound complexes cytochrome b6f and photosystem I. The homologous complex between cytochrome f and plastocyanin, both from spinach, is the object of this study. The solution structure of the reduced spinach plastocyanin was determined using high field NMR spectroscopy, whereas the model structure of oxidized cytochrome f was obtained by homology modeling calculations and molecular dynamics. The model structure of the intermolecular complex was calculated using the program AUTODOCK, taking into account biological information obtained from mutagenesis experiments. The best electron transfer pathway from the heme group of cytochrome f to the copper ion of plastocyanin was calculated using the program HARLEM, obtaining a coupling decay value of 1.8 x 10(-4). Possible mechanisms of interaction and electron transfer between plastocyanin and cytochrome f were discussed considering the possible formation of a supercomplex that associates one cytochrome b6f, one photosystem I, and one plastocyanin.

Global Analysis of Steady-State Energy Transfer Measurements in Membranes: Resolution of Structural and Binding Parameters

A method has been developed allowing structural and binding parameters to be recovered by global analysis of two-dimensional array of steady-state RET data in the special case where energy acceptors distribute between aqueous and lipid phases while donors are embedded in the membrane at a known depth. To test the validity of this approach, correlation and error analyses have been performed using simulated data. To exemplify the method application to the membrane studies, energy transfer from anthrylvinyl-labeled phosphatidylcholine incorporated into mixed phosphatidylcholine/cardiolipin unilamellar vesicles to heme group of cytochrome c is analyzed.

Cytochrome c location in phosphatidylcholine/cardiolipin model membranes: resonance energy transfer study

Resonance energy transfer between lipid-bound fluorescent probe 3-methoxybenzanthrone as a donor and heme group of cytochrome c as an acceptor has been examined to ascertain the protein disposition relative to the surface of model membranes composed of phosphatidylcholine and cardiolipin (10, 50 and 80 mol%). The model of energy transfer in membrane systems has been extended to the case of donors distributed between the two-bilayer leaflets and acceptors located at the outer monolayer taking into account the donor and acceptor orientational behavior. Assuming specific protein orientation relative to the membrane surface and varying lateral distance of the donor–acceptor closest approach in the range from 0 to 3.5 nm the limits for possible heme distances from the bilayer midplane have been found to be 0.8–3 nm (10 mol% CL), 0–2.6 nm (50 mol% CL), and 1.4–3.3 nm (80 mol% CL).

The bonding of nitrosonium ion to sulfur in models of cytochrome c-type biomolecules: an ab initio computational study

The possibility that NO+ could bind to one of the two thioether sulfur atoms that covalently link the heme group of cytochrome c to the polypeptide chain, and that the resulting nitrososulfonium ion might undergo S–C cleavage to form a carbocation and a nitrosothiol were investigated computationally, by HF/3-21G(∗) geometry optimizations. The molecules studied were relatively small cytochrome model compounds with only the iron(II) porphyrin unit, and histidine and Me2S molecules coordinated as axial ligands to the heme iron, and R–S+(NO)–CHMe–porphyrin (R=cysteine–histidine, 1; n-butyl, 2; n-pentyl, 3; methyl, 4Me; n-propyl, 4Pr; CH2CH(NH2)CO–NH–CH(Me)–COOH, 5; and 1 without the iron and the Me2S, 6. Molecules 1, 2, 3 and 5 were stationary points on the potential energy surface (they exist at the HF/3-21G(∗) level); on attempted optimization of 4 and 6 the S–CHMe bond breaks tending to give a nitrosothiol RSNO and a delocalized cation +CHMe–porphyrin. This behavior was rationalized by idealizing the iron porphyrin moiety as Fe++ and porphyrin−−; electron density on the porphyrin ring, when not drained away into the iron, can assist with expulsion of the RSNO leaving group (equivalently, departure of RSNO gives a highly delocalized cation +CHMe–porphyrin). Besides iron, a minimum size for R (n-C4H9 or bigger) is needed to hold R in place, perhaps as a manifestation of the ponderal effect. From calculations on smaller molecules, rough CBS-4 water solvation-corrected reaction energies were estimated for loss of NO+ from 1, 2, and 3 (171, 154 and 154kJmol−1, respectively; binding is thermodynamically favored), and for S–CHMe bond breaking of 2 and 3 (−63 and −63kJmol−1, respectively; cleavage is thermodynamically favored).

Charge Distribution of Cytochrome c Monolayer Using Electrostatic Force Microscope

The charge trapping of the cytochrome c was investigated by electrostatic force microscopy (EFM). The cytochrome c was modified by SPDP and deposited onto the gold substrate by self-assembly technique. The charge was transported to heme groups of cytochrome c by scanning tunneling microscopy (STM) tip, and it was verified that the charge was trapped onto the cytochrome c film surface by EFM. The charge distribution on the cytochrome c surface was observed by EFM measurement.

Charge Distribution of Cytochrome c Monolayer Using Electrostatic Force Microscope

The charge trapping of the cytochrome c was investigated by electrostatic force microscopy (EFM). The cytochrome c was modified by SPDP and deposited onto the gold substrate by self-assembly technique. The charge was transported to heme groups of cytochrome c by scanning tunneling microscopy (STM) tip, and it was verified that the charge was trapped onto the cytochrome c film surface by EFM. The charge distribution on the cytochrome c surface was observed by EFM measurement.

Dissociation reactions of gaseous ferro-, ferri-, and apo-cytochrome c ions

Electrochemical reduction of the iron bound in the heme group of cytochrome c is shown to occur in the nano-electrospray capillary if the protein is sprayed from neutral water using a steel wire as the electrical contact. Quadrupole ion trap collisional activation is used to study the dissociation reactions of cytochrome c as a function of the oxidation state of the iron. Oxidized (Fe(III)) cytochrome c dissociates via sequence-specific amide bond cleavage, while the reduced (Fe(II)) form of the protein dissociates almost exclusively by loss of protonated heme. Apo-cytochrome c, from which the heme has been removed either via gas-phase dissociation of the reduced holo-protein or via solution chemistry, dissociates via amide bond cleavage in similar fashion to the oxidized holo-protein.

Theoretical Study of Trapping Sites for Muon in the Heme Group of Cytochrome c and Associated Shifts in Muon Spin Relaxation Frequencies

Muon Spin Relaxation (μSR) experiments are currently being conducted on the important electron transfer molecule cytochrome c (cyt c) with the goal to find out about microscopic details of the path the moving electron is taking. Simultaneously we are using the Unrestricted Hartree–Fock Cluster Procedure to determine the trapping sites for muon (μ+) and muonium (Mu) in the heme unit of cyt c and the associated hyperfine interactions. For the trapping sites with the highest binding energies for μ+, namely the nitrogen and the carbon of the pyrrole rings, we have used the available magnetic susceptibility data together with our calculated hyperfine fields to predict the Knight-shifts. At room-temperature we found 88.4 ppm and 79.0 ppm for the most attractive N- and adjacent C-site, respectively. At 150 K, these shifts increase to 172.7 ppm for the N-site and 153.7 ppm for the C-site.

Structure of cytochrome c complexes with phospholipids as revealed by resonance energy transfer

Resonance energy transfer between a series of lipid-bound fluorescent probes as donors and the heme group of cytochrome c as acceptor has been used to obtain structural information on the protein complexes with model membranes, composed of phosphatidylcholine and cardiolipin. Analysis of experimental data in terms of the model of energy transfer in two-dimensional systems provides further evidence for preferential cytochrome c orientation with respect to the lipid bilayer and penetration of the protein into the membrane interior.

Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c1.

The reactions of a beef heart cytochrome c1 preparation containing the hinge protein with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) (Ru(II)) were studied by flash photolysis. All of the ruthenium-cytochrome c derivatives formed complexes with cytochrome c1 in low ionic strength buffer (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a 0.4-microseconds laser flash resulted in rapid electron transfer to the ferric heme group in cytochrome c, followed by electron transfer from the ferrous heme group of cytochrome c to the ferric heme group of cytochrome c1. The kinetic difference spectra displayed maxima at 546 nm and minima at 554 nm characteristic of electron transfer between the two cytochromes. The rate constants were independent of concentration at low ionic strength, indicating intracomplex electron transfer. The rate constants were 4,800, 6,800, 22,000, and 22,000 s-1 for cytochrome c derivatives modified at lysines 13, 27, 25, and 72, respectively. The observed rate constants were independent of ionic strength up to about 50 nM and then decreased progressively with further increases in ionic strength indicating dissociation of the complex. Second-order kinetics were observed at 310 mM ionic strength, with rate constants of 1.0 x 10(6), 1.6 x 10(7), 1.2 x 10(8), and 3.0 x 10(7) M-1 s-1 for the derivatives modified at lysines 13, 27, 25, and 72, respectively. The ionic strength dependence of the second-order rate constants is comparable to that involving native horse cytochrome c and is consistent with electron transfer reactions between oppositely charged proteins.

Integrated optical waveguide attenuated total reflection spectrometry and resonance Raman spectroscopy of adsorbed cytochrome c

The heme group of cytochrome c (Cyt C) has resonance absorptions at 520 and 550 nm that arise from x-y degenerate in-plane electronic transitions of the heme moiety. In the present paper, horse heart Cyt C was adsorbed to the surface of a micron-thick silicon oxynitride integrated optical waveguide configurated into a liquid flow cell and prism-coupled with 514.5-nm laser light that was polarized horizontal (TE) and nearly vertical (TM) with respect to the waveguide surface. The adsorbed protein film absorbed light from the evanescent field at the waveguide surface, resulting in two measurable quantities: (1) an increased attenuation of the guided mode intensity in the waveguide and (2) excitation of Cyt C resonance Raman emission

Intracomplex electron transfer between ruthenium-cytochrome c derivatives and cytochrome c oxidase

The reactions of bovine cytochrome c oxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine)ruthenium (II) were studied by laser flash photolysis. All of the derivatives form complexes with cytochrome c oxidase at low ionic strength (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in rapid electron transfer to the ferric heme group of cytochrome c, followed by electron transfer to cytochrome c oxidase. The photoreduced heme Fe(II) in the cytochrome c derivative modified at lysine 25 on the periphery of the heme crevice domain transferred an electron to CuA with a rate constant of 1.1 x 10(4) s-1. CuA then transferred an electron to cytochrome a with a rate constant of 2.3 x 10(4) s-1. The derivatives modified at lysines 7, 39, 55, and 60 remote from the heme crevice domain of cytochrome c have nearly the same kinetics. The rate constant for electron transfer from the cytochrome c heme to CuA is greater than 10(5) s-1, and the rate constant for electron transfer from CuA to cytochrome a is 2 x 10(4) s-1. The cytochrome c derivatives modified at lysines 13 and 27 in the heme crevice domain react much more slowly than the other derivatives, with intracomplex rate constants for oxidation of cytochrome c ranging from 1000 to 6000 s-1. The bulky ruthenium group at the heme crevice domain of these derivatives apparently alters the binding orientation, leading to smaller electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracomplex electron transfer between ruthenium-65-cytochrome b5 and position-82 variants of yeast iso-1-cytochrome c.

We tested the idea that the aromatic ring on the invariant residue Phe-82 in cytochrome c acts as an electron-transfer bridge between cytochrome c and cytochrome b5. Ru-65-cyt b5 was prepared by labeling the single sulfhydryl group on T65C cytochrome b5 with [4-(bromomethyl)-4'-methylbipyridine][bis(bipyridine)]ruthenium 2+ as previously described [Willie, A., Stayton, P.S., Sligar, S.G., Durham, B., & Millett, F. (1992) Biochemistry 31, 7237-7242]. Laser excitation of the complex formed between Ru-65-cyt b5 and Saccharomyces cerevisiae iso-1-cytochrome c at low ionic strength results in rapid electron transfer from the excited-state Ru(II*) to the heme group of Ru-65-cyt b5 followed by biphasic electron transfer to the heme group of cytochrome c with rate constants of (1.0 +/- 0.2) x 10(5) s-1 and (2.0 +/- 0.04) x 10(4) s-1. Variants of iso-1-cytochrome c substituted at Phe-82 with Tyr, Gly, Leu, and Ile have fast-phase rate constants of 0.4, 1.9, 2.1, and 2.0 x 10(5) s-1 and slow-phase rate constants of 5.3, 3.5, 2.4, and 2.0 x 10(3) s-1, respectively. Increasing the ionic strength to 50 mM results in single-phase intracomplex electron transfer with rate constants of 3.8, 3.1, 3.0, 5.0, and 4.5 x 10(4) s-1 for the wild-type, Tyr, Gly, Leu, and Ile variants, respectively. These results demonstrate that an aromatic side chain at residue 82 is not needed for rapid electron transfer with cytochrome b5. Furthermore, two conformational forms of the complex are present at low ionic strength with fast and slow electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Long-distance cofactor interactions in terminal oxidases studied by second-derivative absorption spectroscopy

The electronic transitions of the two heme groups of cytochrome c oxidase have been resolved by application of second-derivative and cryogenic absorption spectroscopy. Both methods reveal a splitting of the ferrocytochrome a Soret transition into two features at 443 and 450 nm. The relative intensity of the 450 nm feature appears to depend on the ligation state of cytochrome a3, the solution pH, and complex formation with cytochrome c. The structural origin and mechanistic significance of this second Soret transition of cytochrome a are discussed in terms of the electron transfer and proton translocation activities of the enzyme.