Peroxidase activity of cytochrome c in its compact state depends on dynamics of the heme region

Cytochrome c (cyt c) is a small globular hemoprotein with the main function as an electron carrier in mitochondrial respiratory chain. Cyt c possesses also peroxidase-like activity in the native state despite its six-coordinated heme iron. In this work, we studied the effect of increasing urea concentration in the range from 0 M to 6 M at pH 7 (pH value of the bulk solvent) and pH 5 (pH value close to negatively charged membrane) on peroxidase-like activity of cyt c. We show that peroxidase-like activity, measured by guaiacol oxidation and the ferrous oxidation in xylenol orange methods, correlates with the accessibility of the heme iron, which was assessed from the association rate constant of cyanide binding to cyt c. Cyt c peroxidase-like activity linearly increases in the pre-denaturational urea concentrations (0–4 M) at both studied pHs without an apparent formation of penta-coordinated state of the heme iron. Our results suggest that dynamic equilibrium among the denaturant-induced non-native coordination states of cyt c, very likely due to reversible unfolding of the least stable foldons, is pre-requisite for enhanced peroxidase-like activity of cyt c in its compact state. Dynamic replacement of the native sixth coordination bond of methionine-80 by lysines (72, 73, and 79) and partially also by histidines (26 and 33) provides an efficient way how to increase peroxidase-like activity of cyt c without significant conformational change at physiological conditions.     

Structure analysis and characterization of the cytochrome c-554 from thermophilic green sulfur photosynthetic bacterium Chlorobaculum tepidum

The cytochrome (Cyt) c-554 in thermophilic green photosynthetic bacterium Chlorobaculum tepidum serves as an intermediate electron carrier, transferring electrons to the membrane-bound Cyt c _z from various enzymes involved in the oxidations of sulfide, thiosulfate, and sulfite compounds. Spectroscopically, this protein exhibits an asymmetric α-absorption band for the reduced form and particularly large paramagnetic ¹H NMR shifts for the heme methyl groups with an unusual shift pattern in the oxidized form. The crystal structure of the Cyt c-554 has been determined at high resolution. The overall fold consists of four α-helices and is characterized by a remarkably long and flexible loop between the α3 and α4 helices. The axial ligand methionine has S-chirality at the sulfur atom with its C^εH₃ group pointing toward the heme pyrrole ring I. This configuration corresponds to an orientation of the lone-pair orbital of the sulfur atom directed at the pyrrole ring II and explains the lowest-field ¹H NMR shift arising from the 18¹ heme methyl protons. Differing from most other class I Cyts c, no hydrogen bond was formed between the methionine sulfur atom and polypeptide chain. Lack of this hydrogen bond may account for the observed large paramagnetic ¹H NMR shifts of the heme methyl protons. The surface-exposed heme pyrrole ring II edge is in a relatively hydrophobic environment surrounded by several electronically neutral residues. This portion is considered as an electron transfer gateway. The structure of the Cyt c-554 is compared with those of other Cyts c, and possible interactions of this protein with its electron transport partners are discussed.     

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.     

In situ monitoring of the catalytic activity of cytochrome C oxidase in a biomimetic architecture

Cytochrome c oxidase (CcO) from Paracoccus denitrificans was immobilized in a strict orientation via a his-tag attached to subunit I on a gold film and reconstituted in situ into a protein-tethered bilayer lipid membrane. In this orientation, the cytochrome c (cyt c) binding site is directed away from the electrode pointing to the outer side of the protein-tethered bilayer lipid membrane architecture. The CcO can thus be activated by cyt c under aerobic conditions. Catalytic activity was monitored by impedance spectroscopy, as well as cyclic voltammetry. Cathodic and anodic currents of the CcO with cyt c added to the bulk solution were shown to increase under aerobic compared to anaerobic conditions. Catalytic activity was considered in terms of repeated electrochemical oxidation/reduction of the CcO/cyt c complex in the presence of oxygen. The communication of cyt c bound to the CcO with the electrode is discussed in terms of a hopping mechanism through the redox sites of the enzyme. Simulations supporting this hypothesis are included.     

Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c₅₅₂ forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c₅ exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c₅₅₂ and Pseudomonas aeruginosa (PA) cyt c₅₅₁ domain-swapped dimers. Positively charged horse cyt c and HT cyt c₅₅₂ domain swapped during expression in E. coli, whereas negatively charged PA cyt c₅₅₁ and SV cyt c₅ did not. Oligomers were formed during expression in E. coli for HT cyt c₅₅₂ attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c₅₅₁ attached to either signal peptide. HT cyt c₅₅₂ formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c₅₅₂ by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c₅₅₁ and SV cyt c₅ did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.     

Cytochrome c-Lipid Interactions: New Insights from Resonance Energy Transfer

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.     

Lytic and Non-Lytic Permeabilization of Cardiolipin-Containing Lipid Bilayers Induced by Cytochrome c

The release of cytochrome c (cyt c) from mitochondria is an important early step during cellular apoptosis, however the precise mechanism by which the outer mitochondrial membrane becomes permeable to these proteins is as yet unclear. Inspired by our previous observation of cyt c crossing the membrane barrier of giant unilamellar vesicle model systems, we investigate the interaction of cyt c with cardiolipin (CL)-containing membranes using the innovative droplet bilayer system that permits electrochemical measurements with simultaneous microscopy observation. We find that cyt c can permeabilize CL-containing membranes by induction of lipid pores in a dose-dependent manner, with membrane lysis eventually observed at relatively high (µM) cyt c concentrations due to widespread pore formation in the membrane destabilizing its bilayer structure. Surprisingly, as cyt c concentration is further increased, we find a regime with exceptionally high permeability where a stable membrane barrier is still maintained between droplet compartments. This unusual non-lytic state has a long lifetime (>20 h) and can be reversibly formed by mechanically separating the droplets before reforming the contact area between them. The transitions between behavioural regimes are electrostatically driven, demonstrated by their suppression with increasing ionic concentrations and their dependence on CL composition. While membrane permeability could also be induced by cationic PAMAM dendrimers, the non-lytic, highly permeable membrane state could not be reproduced using these synthetic polymers, indicating that details in the structure of cyt c beyond simply possessing a cationic net charge are important for the emergence of this unconventional membrane state. These unexpected findings may hold significance for the mechanism by which cyt c escapes into the cytosol of cells during apoptosis.     

Soft perforation of cardiolipin-containing planar lipid bilayer membrane by cytochrome c and H2O2

The release of cytochrome c (cyt c) from mitochondria is responsible for initiation of cell apoptosis. Although extramitochondrial proteins are thought to initiate this release, the exact mechanism remains unclear. Cyt c binds to and penetrates lipid bilayer membranes of specific phospholipid cardiolipin (CL) contained in mitochondria. We present here the experimental results of monitoring planar BLM (pBLM) from mixtures of azolectin and of CL (4/1 by moles) by triangle voltage pulses of 100 mV in amplitude and frequency of 2 Hz. The BLM were modified by a successive addition of cyt c and of H₂O₂ in water solution. It is shown that the addition of cyt c alone leads to a stepwise increase in the ionic conductance of the pBLM, indicating the appearance of transmembrane pores. Pore lifetimes then reached several seconds at an average pore diameter of ~2 nm. Current–voltage characteristics were then linear and passed through the origin which is characteristic for broad, nonselective ion pores. Subsequent addition of H₂O₂ caused a dramatic increase in transmembrane current at retention of average pore size constant. Observed increase in membrane current is due to growth of a number of pores in an open state. We suggest that hydrogen peroxide in the presence of cyt c promotes a peroxidation of membrane phospholipids to form lysolipids, the embedding of which stabilizes the edge of the pore and the surface of lipid bilayer.     

Electron flow into cytochrome c coupled with reactive oxygen species from the electron transport chain converts cytochrome c to a cardiolipin peroxidase: role during ischemia–reperfusion

Background

Cytochrome c (Cyt c) is a mobile component of the electron transport chain (ETC.) which contains a tightly coordinated heme iron. In pathologic settings, a key ligand of the cyt c's heme iron, methionine (Met₈₀), is oxidized allowing cyt c to participate in reactions as a peroxidase with cardiolipin as a target. Myocardial ischemia (ISC) results in ETC. blockade and increased production of reactive oxygen species (ROS). We hypothesized that during ischemia–reperfusion (ISC-REP); ROS generation coupled with electron flow into cyt c would oxidize Met₈₀ and contribute to mitochondrial-mediated ETC. damage.

Methods

Mitochondria were incubated with specific substrates and inhibitors to test the contributions of ROS and electron flow into cyt c. Subsequently, cyt c and cardiolipin were analyzed. To test the pathophysiologic relevance, mouse hearts that underwent ISC-REP were tested for methionine oxidation in cyt c.

Results

The combination of substrate/inhibitor showed that ROS production and electron flux through cyt c are essential for the oxidation of methionine residues that lead to cardiolipin depletion. The content of cyt c methionine oxidation increases following ISC-REP in the intact heart.

Conclusions

Increase in intra-mitochondrial ROS coupled with electron flow into cyt c, oxidizes cyt c followed by depletion of cardiolipin. ISC-REP increases methionine oxidation, supporting that cyt c peroxidase activity can form in the intact heart.

General significance

This study identifies a new site in the ETC. that is damaged during cardiac ISC-REP. Generation of a neoperoxidase activity of cyt c favors the formation of a defective ETC. that activates signaling for cell death.     

Dynamic structure of bombolitin II bound to lipid bilayers as revealed by solid-state NMR and molecular-dynamics simulation

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state ³¹P and ¹³C NMR spectroscopy. ³¹P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (T_c) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the T_c. ¹³C NMR spectra of site-specifically ¹³C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers.     

Domain-Swapped Dimer of Pseudomonas aeruginosa Cytochrome c 551: Structural Insights into Domain Swapping of Cytochrome c Family Proteins

Cytochrome c (cyt c) family proteins, such as horse cyt c, Pseudomonas aeruginosa cytochrome c ₅₅₁ (PA cyt c ₅₅₁), and Hydrogenobacter thermophilus cytochrome c ₅₅₂ (HT cyt c ₅₅₂), have been used as model proteins to study the relationship between the protein structure and folding process. We have shown in the past that horse cyt c forms oligomers by domain swapping its C-terminal helix, perturbing the Met–heme coordination significantly compared to the monomer. HT cyt c ₅₅₂ forms dimers by domain swapping the region containing the N-terminal α-helix and heme, where the heme axial His and Met ligands belong to different protomers. Herein, we show that PA cyt c ₅₅₁ also forms domain-swapped dimers by swapping the region containing the N-terminal α-helix and heme. The secondary structures of the M61A mutant of PA cyt c ₅₅₁ were perturbed slightly and its oligomer formation ability decreased compared to that of the wild-type protein, showing that the stability of the protein secondary structures is important for domain swapping. The hinge loop of domain swapping for cyt c family proteins corresponded to the unstable region specified by hydrogen exchange NMR measurements for the monomer, although the swapping region differed among proteins. These results show that the unstable loop region has a tendency to become a hinge loop in domain-swapped proteins.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

The α-helical membrane spanning domain of cytochrome b5 interacts with cytochrome P450 via nonspecific interactions

Cytochrome b₅ (cyt b₅) is an amphipathic membrane-bound heme protein found in the endoplasmic reticulum of eukaryotes. It consists of three domains, an N-terminal cytosolic, hydrophilic domain containing the heme, a short flexible linker and an α-helical membrane-spanning domain. This study investigated whether there are specific side chain helix–helix packing interactions between the COOH-terminal membrane anchor of cyt b₅ and cytochrome P450 (cyt P450) 2B4 in a purified reconstituted system. Alanine was inserted at six positions in the membrane anchor of cyt b₅. Insertion of alanine into an α-helix causes all amino acids at its carboxyl terminus to be rotated by 100°. The ability of the alanine insertion mutants of cyt b₅ to bind to cyt P450 2B4 was similar to that of the wild-type protein as was the ability of the mutant cyts b₅ to stimulate the metabolism of the anesthetic, methoxyflurane. These results demonstrate that the C-terminal hydrophobic α-helix of cyt b₅ does not interact with cyt P450 2B4 through a specific stereochemical fit of amino acid side chains, but rather through nonspecific interactions.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Peroxidative permeabilization of liposomes induced by cytochrome c/cardiolipin complex

Interaction of cytochrome c with mitochondrial cardiolipin converting this electron transfer protein into peroxidase is accepted to play an essential role in apoptosis. Cytochrome c/cardiolipin peroxidase activity was found here to cause leakage of carboxyfluorescein, sulforhodamine B and 3-kDa (but not 10-kDa) fluorescent dextran from liposomes. A marked decrease in the amplitude of the autocorrelation function was detected with a fluorescence correlation spectroscopy setup upon incubation of dye-loaded cardiolipin-containing liposomes with cytochrome c and H₂O₂, thereby showing release of fluorescent markers from liposomes. The cytochrome c/H₂O₂-induced liposome leakage was suppressed upon increasing the ionic strength, in contrast to the leakage provoked by Fe/ascorbate, suggesting that the binding of cyt c to negatively-charged membranes was required for the permeabilization process. The cyt c/H₂O₂-induced liposome leakage was abolished by cyanide presumably competing with H₂O₂ for coordination with the central iron atom of the heme in cyt c. The cytochrome c/H₂O₂ permeabilization activity was substantially diminished by antioxidants (trolox, butylhydroxytoluene and quercetin) and was precluded if fully saturated tetramyristoyl-cardiolipin was substituted for bovine heart cardiolipin. These data favor the involvement of oxidized cardiolipin molecules in membrane permeabilization resulting from cytochrome c/cardiolipin peroxidase activity. In agreement with previous observations, high concentrations of cyt c induced liposome leakage in the absence of H₂O₂, however this process was not sensitive to antioxidants and cyanide suggesting direct membrane poration by the protein without the involvement of lipid peroxidation.     

Crystal Structure of the Electron Carrier Domain of the Reaction Center Cytochrome cz Subunit from Green Photosynthetic Bacterium Chlorobium tepidum

In green sulfur photosynthetic bacteria, the cytochrome c_z (cyt c_z) subunit in the reaction center complex mediates electron transfer mainly from menaquinol/cytochrome c oxidoreductase to the special pair (P840) of the reaction center. The cyt c_z subunit consists of an N-terminal transmembrane domain and a C-terminal soluble domain that binds a single heme group. The periplasmic soluble domain has been proposed to be highly mobile and to fluctuate between oxidoreductase and P840 during photosynthetic electron transfer. We have determined the crystal structure of the oxidized form of the C-terminal functional domain of the cyt c_z subunit (C-cyt c_z) from thermophilic green sulfur bacterium Chlorobium tepidum at 1.3-Å resolution. The overall fold of C-cyt c_z consists of four α-helices and is similar to that of class I cytochrome c proteins despite the low similarity in their amino acid sequences. The N-terminal structure of C-cyt c_z supports the swinging mechanism previously proposed in relation with electron transfer, and the surface properties provide useful information on possible interaction sites with its electron transfer partners. Several characteristic features are observed for the heme environment: These include orientation of the axial ligands with respect to the heme plane, surface-exposed area of the heme, positions of water molecules, and hydrogen-bond network involving heme propionate groups. These structural features are essential for elucidating the mechanism for regulating the redox state of cyt c_z.     

Polyethylene glycol promotes autoxidation of cytochrome c

Cytochrome c (Cyt c) was rapidly oxidized by molecular oxygen in the presence, but not absence of PEG. The redox potential of heme c was determined by the potentiometric titration to be +236?±?3?mV in the absence of PEG, which was negatively shifted to +200?±?4?mV in the presence of PEG. The underlying the rapid oxidation was explored by examining the structural changes in Cyt c in the presence of PEG using UV–visible absorption, circular dichroism, resonance Raman, and fluorescence spectroscopies. These spectroscopic analyses suggested that heme oxidation was induced by a modest tertiary structural change accompanied by a slight shift in the heme position (<1.0?Å) rather than by partial denaturation, as is observed in the presence of cardiolipin. The near-infrared spectra showed that PEG induced dehydration from Cyt c, which triggered heme displacement. The primary dehydration site was estimated to be around surface-exposed hydrophobic residues near the heme center: Ile81 and Val83. These findings and our previous studies, which showed that hydrated water molecules around Ile81 and Val83 are expelled when Cyt c forms a complex with CcO, proposed that dehydration of these residues is functionally significant to electron transfer from Cyt c to CcO.     

Probing the Transmembrane Structure and Dynamics of Microsomal NADPH-cytochrome P450 oxidoreductase by Solid-State NMR

NADPH-cytochrome P450 oxidoreductase (CYPOR) is an essential redox partner of the cytochrome P450 (cyt P450) superfamily of metabolic enzymes. In the endoplasmic reticulum of liver cells, such enzymes metabolize ∼75% of the pharmaceuticals in use today. It is known that the transmembrane domain of CYPOR plays a crucial role in aiding the formation of a complex between CYPOR and cyt P450. Here we present the transmembrane structure, topology, and dynamics of the FMN binding domain of CYPOR in a native membrane-like environment. Our solid-state NMR results reveal that the N-terminal transmembrane domain of CYPOR adopts an α-helical conformation in the lipid membrane environment. Most notably, we also show that the transmembrane helix is tilted ∼13° from the lipid bilayer normal, and exhibits motions on a submillisecond timescale including rotational diffusion of the whole helix and fluctuation of the helical director axis. The approaches and the information reported in this study would enable further investigations on the structure and dynamics of the full-length NADPH-cytochrome P450 oxidoreductase and its interaction with other membrane proteins in a membrane environment.     

Effect of cytochrome c on the phase behavior of charged multicomponent lipid membranes

We studied the effect of submicromolar concentrations of cytochrome c (cyt c) on the phase behavior of ternary lipid membranes composed of charged dioleoylphosphatidylglycerol, egg sphingomyelin and cholesterol. The protein was found to induce micron-sized domains in membranes belonging to the single-fluid-phase region of the protein-free ternary mixture and, as a result, to expand the region of coexistence of liquid ordered (L_o) and liquid disordered (L_d) phases. Direct observations on individual vesicles revealed that protein adsorption increases the area of L_d domains. Measurements using a fluorescent analog of cyt c showed that the protein preferentially adsorbs onto domains belonging to the L_d phase. The adsorption was quantitatively characterized in terms of partitioning ratios between the L_d and the L_o phases. The protein was also found to induce vesicle leakage even at relatively low concentrations. In eukaryotic cells under normal physiological conditions, cyt c is localized within the intermembrane space of mitochondria. During cell apoptotis, cyt c is released into the cytosol and its adsorption to intracellular membranes may strongly perturb the lipid distribution within these membranes as suggested by our results.     

Nitric Oxide Binds to the Proximal Heme Coordination Site of the Ferrocytochrome c/Cardiolipin Complex: FORMATION MECHANISM AND DYNAMICS*

Mammalian mitochondrial cytochrome c interacts with cardiolipin to form a complex (cyt. c/CL) important in apoptosis. Here we show that this interaction leads to structural changes in ferrocytochrome c that leads to an open coordinate site on the central iron, resulting from the dissociation of the intrinsic methionine residue, where NO can rapidly bind (k = 1.2 × 10⁷ m⁻¹ s⁻¹). Accompanying NO binding, the proximal histidine dissociates leaving the heme pentacoordinate, in contrast to the hexacoordinate nitrosyl adducts of native ferrocytochrome c or of the protein in which the coordinating methionine is removed by chemical modification or mutation. We present the results of stopped-flow and photolysis experiments that show that following initial NO binding to the heme, there ensues an unusually complex set of kinetic steps. The spectral changes associated with these kinetic transitions, together with their dependence on NO concentration, have been determined and lead us to conclude that NO binding to cyt. c/CL takes place via an overall scheme comparable to that described for cytochrome c′ and guanylate cyclase, the final product being one in which NO resides on the proximal side of the heme. In addition, novel features not observed before in other heme proteins forming pentacoordinate nitrosyl species, include a high yield of NO escape after dissociation, rapid (<1 ms) dissociation of proximal histidine upon NO binding and its very fast binding (60 ps) after NO dissociation, and the formation of a hexacoordinate intermediate. These features all point at a remarkable mobility of the proximal heme environment induced by cardiolipin.