A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

Photo-oxidation of membrane-bound and soluble cytochromec in the green sulfur bacteriumChlorobium tepidum

We studied the photosynthetic electron transfer system of membrane-bound and soluble cytochromec inChlorobium tepidum, a thermophilic green sulfur bacterium, using whole cells and membrane preparations. Sulfide and thiosulfate, physiological electron donors, enhanced flash-induced photo-oxidation ofc-type cytochromes in whole cells. In membranes,c-553 cytochromes with two (or three) heme groups served as immediate electron donors for photo-oxidized bacteriochlorophyll (P840) in the reaction center, and appeared to be closely associated with the reaction center complex. The membrane-bound cytochromec-553 had anE _m-value of 180 mV. When isolated soluble cytochromec-553, which has an apparent molecular weight of 10 kDa and seems to correspond to the cytochromec-555 inChlorobium limicola andChlorobium vibrioforme, was added to a membrane suspension, rapid photo-oxidation of both soluble and membrane-bound cytochromesc-553 was observed. The oxidation of soluble cytochromec-553 was inhibited by high salt concentrations. In whole cells, photo-oxidation was observed in the absence of exogenous electron donors and re-reduction was inhibited by stigmatellin, an inhibitor of the cytochromebc complex. These results suggest that the role of membrane-bound and soluble cytochromec inC. tepidum is similar to the role of cytochromec in the photosynthetic electron transfer system of purple bacteria.     

C1 metabolism inParacoccus denitrificans: Genetics ofParacoccus denitrificans

Paracoccus denitrificans is able to grow on the C₁ compounds methanol and methylamine. These compounds are oxidized to formaldehyde which is subsequently oxidized via formate to carbon dioxide. Biomass is produced by carbon dioxide fixation via the ribulose biphosphate pathway. The first oxidation reaction is catalyzed by the enzymes methanol dehydrogenase and methylamine dehydrogenase, respectively. Both enzymes contain two different subunits in an ₂₂ configuration. The genes encoding the subunits of methanol dehydrogenase (moxF andmoxI) have been isolated and sequenced. They are located in one operon together with two other genes (moxJ andmoxG) in the gene ordermoxFJGI. The function of themoxJ gene product is not yet known.MoxG codes for a cytochromec _551i , which functions as the electron acceptor of methanol dehydrogenase. Both methanol dehydrogenase and methylamine dehydrogenase contain PQQ as a cofactor. These so-called quinoproteins are able to catalyze redox reactions by one-electron steps. The reaction mechanism of this oxidation will be described. Electrons from the oxidation reaction are donated to the electron transport chain at the level of cytochromec. P. denitrificans is able to synthesize at least 10 differentc-type cytochromes. Five could be detected in the periplasm and five have been found in the cytoplasmic membrane. The membrane-bound cytochromec ₁ and cytochromec ₅₅₂ and the periplasmic-located cytochromec ₅₅₀ are present under all tested growth conditions. The cytochromesc _551i andc _553i , present in the periplasm, are only induced in cells grown on methanol, methylamine, or choline. The otherc-type cytochromes are mainly detected either under oxygen limited conditions or under anaerobic conditions with nitrate as electron acceptor or under both conditions. An overview including the induction pattern of allP. denitrificans c-type cytochromes will be given. The genes encoding cytochromec ₁, cytochromec ₅₅₀, cytochromec _551i , and cytochromec _553i have been isolated and sequenced. By using site-directed mutagenesis these genes were mutated in the genome. The mutants thus obtained were used to study electron transport during growth on C₁ compounds. This electron transport has also been studied by determining electron transfer rates inin vitro experiments. The exact pathways, however, are not yet fully understood. Electrons from methanol dehydrogenase are donated to cytochromec _551i . Further electron transport is either via cytochromec ₅₅₀ or cytochromec _553i to cytochromeaa ₃. However, direct electron transport from cytochromec _551i to the terminal oxidase might be possible as well. Electrons from methylamine dehydrogenase are donated to amicyanin and then via cytochromec ₅₅₀ to cytochromeaa ₃, but other routes are used also.P. denitrificans is studied by several groups by using a genetic approach. Several genes have already been cloned and sequenced and a lot of mutants have been isolated. The development of a host/vector system and several techniques for mutation induction that are used inP. denitrificans genetics will be described.     

Lumenal proteins involved in respiratory electron transport in the cyanobacterium Synechocystis sp. PCC6803

Cyanobacterial thylakoids catalyze both photosynthetic and respiratory activities. In a photosystem I-less Synechocystis sp. PCC 6803 strain, electrons generated by photosystem II appear to be utilized by cytochrome oxidase. To identify the lumenal electron carriers (plastocyanin and/or cytochromes c ₅₅₃, c ₅₅₀, and possibly c _M) that are involved in transfer of photosystem II-generated electrons to the terminal oxidase, deletion constructs for genes coding for these components were introduced into a photosystem I-less Synechocystis sp. PCC 6803 strain, and electron flow out of photosystem II was monitored in resulting strains through chlorophyll fluorescence yields. Loss of cytochrome c ₅₅₃ or plastocyanin, but not of cytochrome c ₅₅₀, decreased the rate of electron flow out of photosystem II. Surprisingly, cytochrome c _M could not be deleted in a photosystem I-less background strain, and also a double-deletion mutant lacking both plastocyanin and cytochromec ₅₅₃ could not be obtained. Cytochrome c _M has some homology with the cytochrome c-binding regions of the cytochromecaa₃ -type cytochrome oxidase from Bacillus spp. and Thermus thermophilus. We suggest that cytochrome c _M is a component of cytochrome oxidase in cyanobacteria that serves as redox intermediate between soluble electron carriers and the cytochromeaa₃ complex, and that either plastocyanin or cytochrome c ₅₅₃ can shuttle electrons from the cytochrome b_6f complex to cytochrome c _M.     

A further investigation of the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>–cytochrome<Emphasis Type="Italic"> c</Emphasis> complex

The interaction of reduced rabbit cytochrome b₅ with reduced yeast iso-1 cytochrome c has been studied through the analysis of ¹H–¹⁵N HSQC spectra, of ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b₅ has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b₅.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics     

Cellular localization of cytochrome c 553 in the N2-fixing cyanobacterium Anabaena variabilis

The in situ location of the electron carrier protein cytochrome C ₅₅₃ (cyt c ₅₅₃) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a post-ernbedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c ₅₅₃ specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c ₅₅₃ during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabilis. The results indicate that most of cyt c ₅₅₃ is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.Abbreviations Cyt c ₅₅₃ cytochrome c ₅₅₃ - PBS phosphate buffered saline (20 mM sodium phosphate, 0.9% NaCl, pH 7.4) - PMSF phenylmethylsulfonyl fluoride Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz.     

Orientation of complex III in the yeast mitochondrial membrane: Labeling with [125I] diazobenzenesulfonate and functional studies with the decyl analogue of coenzyme Q as substrate

Mitochondria (or mitoplasts) and submitochondrial particles from yeast were treated with [¹²⁵I] diazobenzenesulfonate to label selectively proteins exposed on the outer or inner surface of the inner mitochondrial membrane. Polyacrylamide gel analysis of the immunoprecipitates formed with antibodies against Complex III or cytochromeb revealed that the two core proteins and cytochromeb were labeled in both mitochondria and submitochondrial particles, suggesting that these proteins span the membrane. Cytochromec ₁ and the iron sulfur protein were labeled in mitochondria but not in submitochondrial particles, suggesting that these proteins are exposed on the cytosolic side of the inner membrane. The steady-state reduction of cytochromesb andc ₁ was determined with succinate and the decyl analogue of coenzyme Q as substrates. Addition of the coenzyme Q analogue to mitochondria caused reduction of 15–30% of the total dithionite-reducibleb and 100% of the cytochromec ₁: Addition of the coenzyme Q analogue to submitochondrial particles led to the reduction of 70% of the total dithionite-reducible cytochromeb but insignificant amounts of cytochromec ₁. A model to explain the topography of Complex III in the inner membrane is proposed based on these results.Abbreviations used: DABS, diazobenzene sulfonate; DBH₂, reduced form of decyl analogue of coenzyme Q (2,3-dimethoxy-5-methyl-6-n-decyl-1,4-benzoquinone); PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.     

Interactions involving the cyanine dye,diS-C3-(5), cytochromec and liposomes and their implications for estimations of Δψ in cytochromec oxidase-reconstituted proteoliposomes

Summary The interference of cytochromec with absorption and fluorescence changes of the cyanine dye, diS-C₃-(5), was investigated in the presence of liposomes and cytochromec-oxidase reconstituted proteoliposomes. The apparent cytochromec-dependent quenching of diS-C₃-(5) fluorescence, and the associated absorbance losses in the presence of liposomes and proteoliposomes in low ionic strength media, are due to destruction of the dye caused by cytochromec-mediated lipid peroxidation. The rate of this reaction was further enhanced in the presence of hydrogen peroxide. Even in the absence of liposomes or proteoliposomes, a cytochromec-induced breakdown of dye was observed in the presence of hydrogen peroxide.The cytochromec mediated absorbance and fluorescence losses of diS-C₃-(5) in liposomal or proteoliposomal systems are prevented by Ca²⁺ and La³⁺ ions, by ascorbate, by high ionic strength and by the antioxidant BHT. Under these conditions, the process of lipid peroxidation mediated by cytochromec is inhibited either directly (e. g. by BHT) or indirectly, by preventing the binding of cytochromec to lipid vesicles. The impact of these findings upon the experimental estimation of membrane potential inaa ₃-reconstituted proteoliposomes is discussed.     

The Temporal Relationship between Protein Phosphatase, Mitochondrial CytochromecRelease, and Caspase Activation in Apoptosis

Apoptosis is mediated by members of the caspase family of proteases which can be activated by release of mitochondrial cytochromec.Additional members of the caspase family are activated at the cell surface in response to direct stimulus from the external environment such as by activation of the Fas receptor. It has been suggested that these upstream caspases directly activate the downstream caspases which would obviate a role for cytochromecin apoptosis induced by the Fas receptor. We demonstrate that cytochromecis released from mitochondria of Jurkat cells in response to both staurosporine and an agonistic anti-Fas antibody and that only the latter is inhibited by the caspase inhibitor z-VAD-FMK. This suggests that an upstream caspase such as caspase-8 is required for the Fas-mediated release of mitochondrial cytochromec.The protein phosphatase inhibitor calyculin A prevented cytochromecrelease and apoptosis induced by both agents, suggesting that release of cytochromecis required in both models. Zinc, once thought of as an endonuclease inhibitor, has previously been shown to prevent the activation of caspase-3. We show that zinc prevents the activation of downstream caspases and apoptosis induced by both insults, yet does not prevent release of mitochondrial cytochromec.The ability of calyculin A and zinc to prevent DNA digestion implies that the mitochondrial pathway is important for induction of apoptosis by both agents. These results do not support an alternative pathway in which caspase-8 directly activates caspase-3. These results also demonstrate that a critical protein phosphatase regulates the release of cytochromecand apoptosis induced by both insults.     

Interaction of cytochromec with phospholipid monolayers and bilayer membranes

Summary For the study of the interaction between oxidized cytochromec and phosphatidylinositide, two different model systems were used: (1) monolayers which were deposited after the method of Langmuir and Blodgett onto glass plates, and (2) bimolecular (“black”) membranes in aqueous phase. The amount of bound protein was determined with a sensitive spectrophotometer. It was found that at low ionic strength about 10¹³ cytochromec molecules per cm² are bound to the lipid surface, which nearly corresponds to a densely packed monolayer. At high ionic strength (∼ 0.1m) or low pH (pH<3), the adsorbed protein layer becomes unstable. This result indicates that the interaction is mainly electrostatic. In accordance with this conclusion is the observation that the rate of adsorption is diffusion controlled; i.e., almost every protein molecule hitting the surface is bound. The cytochromec monolayer can be reduced by ascorbate. In contrast to ferrocytochromec in solution, the bound ferrocytochrome was found to be autoxidable.     

Cytochromec oxidase ofEuglena gracilis: Purification,characterization, and identification of mitochondrially synthesized subunits

Cytochromec oxidase was purified from mitochondria ofEuglena gracilis and separated into 15 different polypeptide subunits by polyacrylamide gel electrophoresis. All 15 subunits copurify through various purification procedures, and the subunit composition of the isolated enzyme is identical to that of the immunoprecipitated one. Therefore, the 15 protein subunits represent integral components of theEuglena oxidase. In anin vitro protein-synthesizing system using isolated mitochondria, polypeptides 1–3 were radioactive labeled in the presence of [³⁵S]methionine. This further identifies these polypeptides with the three largest subunits of cytochromec oxidse encoded by mitochondrial DNA in other eukaryotic organisms. By subtraction, the other 12 subunits can be assigned to nuclear genes. The isolatedEuglena oxidase was highly active withEuglena cytochromec ₅₅₈ and has monophasic kinetics. Using horse cytochromec ₅₅₀ as a substrate, activity of the isolated oxidase was rather low. These findings correlate with the oxidase activity of mitochondrial membranes. Again, reactivity was low with cytochromec ₅₅₀ and 35-fold higher with theEuglena cytochromec ₅₅₈. The data show that the cytochromec oxidase of the protistEuglena is different from other eukaryotic cytochromec oxidases in number and size of subunits, and also with regard to kinetic properties and substrate specificity.Abbreviations kDa kilodalton - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TN turnover number     

Protein stability and mutations in the axial methionine loop of a minimal cytochrome<Emphasis Type="Italic"> c</Emphasis>

The minimal mono-heme ferricytochrome c from Bacillus pasteurii, containing 71 amino acids, has been further investigated through mutagenesis of different positions in the loop containing the iron ligand Met71. These mutations have been designed to sample different aspects of the loop structure, in order to obtain insights into the determinants of the stability of the iron(III) environment. In particular, positions 68, 72 and 75 have been essayed. Gln68 has been mutated to Lys to provide a suitable alternate ligand that can displace Met71 under denaturing conditions. Pro72 has been mutated to Gly and Ala to modify the range of allowed backbone conformations. Ile75, which is in van der Waals contact with Met71 and partly shields a long-lived water molecule in a protein cavity, has been substituted by Val and Ala to affect the network of inter-residue interactions around the metal site. The different contributions of the above amino acids to protein parameters such as structure, redox potential and the overall stability against unfolding with guanidinium hydrochloride are analyzed. While the structure remains essentially the same, the stability decreases with mutations. The comparison with mitochondrial c-type cytochromes is instructive.Abbreviations Bpcytc soluble fragment of cytochrome c₅₅₃ from Bacillus pasteurii - GdmCl guanidinium chloride - I75A Ile75 to Ala mutant - I75V Ile75 to Val mutant - P72A Pro72 to Ala mutant - P72G Pro72 to Gly mutant - Q68K Gln75 to Lys mutant - WT wild type     

Simultaneous measurements of optical and electrical properties of artificial membranes composed of mitochondrial lipids and their interaction with cytochromec

Summary A newly constructed cell, which allows simultaneous measurements of optical and electrical properties, was used to study bimolecular black membranes composed of beef heart mitochondrial lipids and their interaction with cytochromec.The results show that these highly charged membranes are stable only in relatively limited ranges of boundary conditions. In 0.1n KCl their maximum direct current (dc) resistance is 7×10⁸ Ohm cm²±10%; the series capacity at 1kHz is 0.43 F/cm²±3%; the entire thickness, determined by optical reflectivity, is 5.8±0.2 nm.The interaction between oxidized cytochromec and these lipid membranes is primarily of electrostatic nature, and dependent on the presence of highly charged phospholipids, such as diphosphatidyl glycerol (cardiolipin) and phosphatidyl ethanolamine. The attachment of cytochromec maximally causes a 2.5-fold increase in reflectivity, without any noticeable change in the capacity. This leads to a subsequent instability of the membrane (i.e., rupture) preceded by a rapid increase of the dc conductivity. This behavior is far less pronounced with reduced cytochromec.     

The K+-ionophores nonactin and valinomycin interact differently with the protein of reconstituted cytochromec oxidase

The K⁺-ionophores valinomycin and nonactin induce a qualitatively identical change of the visible spectrum of isolated oxidized cytochromec oxidase (red shift), but the amplitude is half with nonactin. Valinomycin, in the presence or absence of a protonophore, stimulates the respiration of the reconstituted enzyme to a higher extent than nonactin and results in a higherK _m for cytochromec. In contrast, nonactin causes a fivefold rate of proton conductivity across a liposomal membrane, after induction of a K⁺-diffusion potential. The data indicate that respiratory control by these antibiotics is not only due to degradation of a membrane potential, but rather to specific interaction with and modification of cytochromec oxidase.     

Direct monitoring of the electron pool effect of cytochrome<Emphasis Type="Italic"> c</Emphasis><Subscript>3</Subscript> by highly sensitive EQCM measurements

Cytochrome c₃ from Desulfovibrio vulgaris has four hemes per molecule, and a redox change at the hemes alters the conformation of the protein, leading to a redox-dependent change in the interaction of cytochrome c₃ with redox partners (an electron acceptor or an electron donor). The redox-dependent change in this interaction was directly monitored by the high-performance electrochemical quartz crystal microbalance (EQCM) technique that has been improved to give high sensitivity in solution. In this method, cytochrome c₃ molecules in solution associate electrostatically with a viologen-immobilized quartz crystal electrode as a monolayer, and redox of the associating cytochrome c₃ is controlled by the immobilized viologen. This technique makes it possible to measure the access of cytochrome c₃ to the electrode or repulsion from the electrode, and hence interconversion between an electrostatic complex and an electron transfer complex on the cytochrome c₃ and the viologen as a mass change accompanying a potential sweep is monitored. In addition, simultaneous measurement of a mass change and a potential step reveals that the cytochrome c₃ stores electrons when the four hemes are reduced (an electron pool effect), that is, the oxidized cytochrome c₃ facilitates acceptance of electrons from the immobilized viologen molecule, but the reduced cytochrome c₃ donates the accepted electrons to the viologen with difficulty.     

Overexpression and Purification of CytochromecOxidase fromRhodobacter sphaeroides

Theaa₃-type cytochromecoxidase ofRhodobacter sphaeroideshas been overexpressed up to seven fold over that in wild-type strains by engineering a multicopy plasmid with all the required oxidase genes and by establishing optimum growth conditions. The two operons containing the three structural genes and two assembly genes for cytochromecoxidase were ligated into a pUC19 vector and reintroduced into several oxidase-deletedR. sphaeroidesstrains. Under conditions of relatively high pH and maximal aeration, high levels of expression were observed. A smaller expression vector, pBBR1MCS, and a fructose promoter (fruP)⁵were found not to enhance cytochromecoxidase expression inR. sphaeroides.An improved cytochromecoxidase purification protocol is reported, which combines histidine elution from a nickel affinity column and anion-exchange chromatography, and results in a higher yield and purity than previously obtained.     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Effect of low-molecular-weight proteins on protein (lysozyme) binding to isolated brush-border membranes of rat kidney

Filtered proteins including the low-molecular-weight protein lysozyme are reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with binding of the protein to the brush-border membrane. The binding of ¹²⁵I-labelled egg-white lysozyme (EC 3.2.1.17) to isolated brush-border membranes of rat kidney and the effect of several low-molecular weight proteins on that binding was determined. The Scatchard plot revealed a one-component binding type with a dissociation constant of 5.3 μM and 53.0 nmol/mg membrane protein for the number of binding sites. The binding of the cationic lysozyme was inhibited competitively by the addition of cationic cytochrome c to the incubation medium, while the neutral myoglobin had no effect. The anionic β-lactoglobulin A inhibited the lysozyme binding in a noncompetitive manner. These data suggest that the binding takes place between positively charged groups of the protein molecule and negative sites on the brush-border membrane, and, the competition between the cationic cytochrome c and the cationic lysozyme for the binding sites may be responsible for the inhibitory effect of cytochrome c on renal lysozyme reabsorption. The binding step at the brush-border membrane appears to be cation-selective.     

Mutational analysis of assembly and function of the iron-sulfur protein of the cytochromebc 1 complex inSaccharomyces cerevisiae

The iron-sulfur protein of the cytochromebc ₁ complex oxidizes ubiquinol at center P in the protonmotive Q cycle mechanism, transferring one electron to cytochromec ₁ and generating a low-potential ubisemiquinone anion which reduces the low-potential cytochromeb-566 heme group. In order to catalyze this divergent transfer of two reducing equivalents from ubiquinol, the iron-sulfur protein must be structurally integrated into the cytochromebc ₁ complex in a manner which facilitates electron transfer from the iron-sulfur cluster to cytochromec ₁ and generates a strongly reducing ubisemiquinone anion radical which is proximal to theb-566 heme group. This radical must also be sequestered from spurious reactivities with oxygen and other high-potential oxidants. Experimental approaches are described which are aimed at understanding how the iron-sulfur protein is inserted into center P, and how the iron-sulfur cluster is inserted into the apoprotein.     

A study of the ultrastructural organization of cytochromec-phospholipid membranes as revealed by various experimental treatments

Summary Two different types of cytochromec-phospholipid model membranes have been investigated by electron-microscopy. The preservation of the lamellar structure of the cytochromec plus phospholipid-water system after extraction of 88% phospholipids with aqueous acetone followed by only aldehyde fixation is revealed.Pronase digestion of this type of model membrane leads to removal of 80% of the protein and loss of ordered structures as revealed by aldehyde post-fixation.It is shown that the hydrated isooctane-soluble cytochromec-phospholipid complex retained the lamellar structures after extraction of 75% phospholipids followed by separate aldehyde and osmium tetroxide fixations. Pronase treatment of this type of model membrane leads to removal of 25 to 30% of the protein. After aldehyde fixation of these pronase-treated samples the triple-layered structures are preserved.The structural integrity of both types of model membranes after organic extractions with aldehyde pre- or post-fixation is thought to be the result of cross-linkage of the protein by aldehyde polymers on both surfaces and through lipid bilayers. Possible localization of cytochromec in investigated model membranes is discussed.