Cytochromes c555 from the hyperthermophilic bacterium Aquifex aeolicus (VF5). 1. Characterization of two highly homologous, soluble and membranous, cytochromes c555.

Two distinct class I (monoheme) c-type cytochromes from the hyperthermophilic bacterium Aquifex aeolicus were studied by biochemical and biophysical methods (i.e., optical and EPR spectroscopy, electrochemistry). The sequences of these two heme proteins (encoded by the cycB1 and cycB2 genes) are close to identical (85% identity in the common part of the protein) apart from the presence of an N-terminal stretch of 62 amino acid residues present only in the cycB1 gene. A soluble cytochrome was purified and identified by N-terminal sequencing as the cycB2 gene product. It showed an alpha-peak at 555 nm, an E(m) value of +220 mV, and electron paramagnetic resonance parameters of gz = 2.89, gy = 2.287, and gx = 1.52. A firmly membrane-bound cytochrome characterized by nearly identical properties was detected and attributed to the cycB1 gene product. The very high degree of homology of its N-terminal part to cytochrome c553 from Heliobacterium gestii strongly suggests it to be anchored to the membrane via N-terminally attached lipid molecules. The two heme proteins were named cytochrome c555s (soluble) and cytochrome c555m (membranous). Electron paramagnetic resonance on partially ordered membrane multilayers suggests that the solvent-exposed heme domain of cytochrome c555m is flexible with respect to the membrane plane. Possible functional roles for both cytochromes are discussed.     

Parallel electron donation pathways to cytochrome c(z) in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c(z) bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c(z). It was also shown that the rereduction rate of cytochrome c(z) by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Crystal structure of auracyanin, a "blue" copper protein from the green thermophilic photosynthetic bacterium Chloroflexus aurantiacus

Auracyanin B, one of two similar blue copper proteins produced by the thermophilic green non-sulfur photosynthetic bacterium Chloroflexus aurantiacus, crystallizes in space group P6(4)22 (a=b=115.7 A, c=54.6 A). The structure was solved using multiple wavelength anomalous dispersion data recorded about the CuK absorption edge, and was refined at 1.55 A resolution. The molecular model comprises 139 amino acid residues, one Cu, 247 H(2)O molecules, one Cl(-) and two SO(4)(2-). The final residual and estimated standard uncertainties are R=0.198, ESU=0.076 A for atomic coordinates and ESU=0.05 A for Cu---ligand bond lengths, respectively. The auracyanin B molecule has a standard cupredoxin fold. With the exception of an additional N-terminal strand, the molecule is very similar to that of the bacterial cupredoxin, azurin. As in other cupredoxins, one of the Cu ligands lies on strand 4 of the polypeptide, and the other three lie along a large loop between strands 7 and 8. The Cu site geometry is discussed with reference to the amino acid spacing between the latter three ligands. The crystallographically characterized Cu-binding domain of auracyanin B is probably tethered to the periplasmic side of the cytoplasmic membrane by an N-terminal tail that exhibits significant sequence identity with known tethers in several other membrane-associated electron-transfer proteins.     

Production of a recombinant hybrid hemoflavoprotein: engineering a functional NADH:cytochrome c reductase.

A gene has been constructed coding for a unique fusion protein, NADH:cytochrome c reductase, that comprises the soluble heme-containing domain of rat hepatic cytochrome b(5) as the amino-terminal portion of the protein and the soluble flavin-containing domain of rat hepatic cytochrome b(5) reductase as the carboxyl terminus. The gene has been expressed in Escherichia coli resulting in the highly efficient production of a functional hybrid hemoflavoprotein which has been purified to homogeneity by a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP agarose, and size-exclusion chromatography. The purified protein exhibited a molecular mass of approximately 46 kDa by polyacrylamide gel electrophoresis and 40,875 Da, for the apoprotein, using mass spectrometry which also confirmed the presence of both heme and FAD prosthetic groups. The fusion protein showed immunological cross-reactivity with both anti-rat cytochrome b(5) and anti-rat cytochrome b(5) reductase antibodies indicating the conservation of antigenic determinants from both native domains. Spectroscopic analysis indicated the fusion protein contained both a b-type cytochrome and flavin chromophors with properties identical to those of the native proteins. Amino-terminal and internal amino acid sequencing confirmed the identity of peptides derived from both the heme- and flavin-binding domains with sequences identical to the deduced amino acid sequence. The isolated fusion protein retained NADH:ferricyanide reductase activity (k(cat) = 8.00 x 10(2) s(-1), K(NADH)(m) = 4 microM, K(FeCN(6))(m) = 11 microM) comparable to that of that of native NADH:cytochrome b(5) reductase and also exhibited both NADH:cytochrome c reductase activity (k(cat) = 2.17 x 10(2) s(-1), K(NADH)(m) = 2 microM, K(FeCN(6))(m) = 11 microM, K(Cyt.c)(m) = 1 microM) and NADH:methemoglobin reductase activity (k(cat) = 4.40 x 10(-1) s(-1), K(NADH)(m) = 3 microM, K(mHb)(m) = 47 microM), the latter two activities indicating efficient electron transfer from FAD to heme and retention of physiological function. This work represents the first successful bacterial expression of a soluble, catalytically competent, rat hepatic cytochrome b(5)-cytochrome b(5) reductase fusion protein that retains the functional properties characteristic of the individual heme and flavin domain.     

Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and "logic gate"-like dual functionality

1Cellobiose dehydrogenase is a hemoflavoenzyme that catalyzes the sequential electron-transfer from an electron-donating substrate (e.g. cellobiose) to a flavin center, then to an electron-accepting substrate (e.g. quinone) either directly or via a heme center after an internal electron-transfer from the flavin to heme. We cloned the dehydrogenase from Humicola insolens, which encodes a protein of 761 amino acid residues containing an N-terminal heme domain and a C-terminal flavin domain, and studied how the catalyzed electron transfers are regulated. Based on the correlation between the rate and redox potential, we demonstrated that with a reduced flavin center, the enzyme, as a reductase, could export electron from its heme center by a "outer-sphere" mechanism. With the "resting" flavin center, however, the enzyme could have a peroxidase-like function and import electron to its heme center after a peroxidative activation. The dual functionality of its heme center makes the enzyme a molecular "logic gate", in which the electron flow through the heme center can be switched in direction by the redox state of the coupled flavin center.     

Cytochrome c and c2 binding dynamics and electron transfer with photosynthetic reaction center protein and other integral membrane redox proteins

To further the understanding of the details of c-type cytochrome action as a redox carrier between major electron-transfer proteins, the single-turnover kinetics time course of cytochrome c and cytochrome c2 oxidation by flash-activated photosynthetic reaction center (purified from the bacterium Rhodobacter sphaeroides) has been examined under a wide variety of conditions of concentration, ionic strength, and viscosity with reaction center present in detergent dispersion and phosphatidylcholine proteoliposomes. We find that the three-state model proposed by Overfield and Wraight [Overfield, R. E., & Wraight, C. A. (1980) Biochemistry 19, 3322-3327] is generally sufficient to model the kinetics time course; many similarities are found with the cytochrome c-cytochrome c oxidase interaction in mitochondria. Further, we find the following: (1) Significant "product inhibition" by oxidized cytochrome c (c2) bound to the reaction center is apparent. (2) The viscosity sensitivity of the electron transfer into the reaction center from bound cytochrome c (c2) suggests a physical interpretation of the distal state. (3) The exchange dynamics of oxidized and reduced cytochrome c (c2) are similar regardless of the state of activation of the reaction center. (4) Preferential binding of the oxidized form of cytochrome c is revealed upon reconstitution of the reaction center into neutral lipid vesicles, permitting an independent confirmation of the binding suggested by the kinetics. (5) Flash-activated electron-transfer kinetics in reaction center hybrid protein systems have shown that diffusion and competitive binding characterize the behavior of cytochrome c as a redox carrier between the reaction center protein and either the cytochrome bc1 complex or the cytochrome c oxidase.     

Structural and functional studies on DHC, the diheme cytochrome c from Rhodobacter sphaeroides, and its interaction with SHP, the sphaeroides heme protein

The diheme cytochrome c (DHC) from Rhodobacter sphaeroides is a soluble protein with a mass of 16 kDa that represents a new class of c-type cytochrome [Vandenberghe, I., et al. (1998) Biochemistry 37, 13075-13081]. The gene encoding DHC is associated with another encoding a cytochrome known as SHP (sphaeroides heme protein). It is believed that DHC is the electron donor for SHP, which is known to bind oxygen. To gain further insight into the properties and role of DHC, we have carried out structure-function studies on the protein and examined its interaction with SHP. The crystal structures of native and recombinant DHC have been determined to resolutions of 1.85 and 2.0 A, respectively. The structures show that DHC folds into two distinct domains each containing one heme. While the N-terminal domain is a class I cytochrome c, the C-terminal domain shows no similarity to any existing structures and thus constitutes a novel cytochrome c structural motif. The shortest, edge-to-edge, distance between the heme groups is 10.2 A, and this distance is bridged by Tyr31, thus ensuring fast internal electron transfer. DHC binds strongly to its proposed physiological partner, SHP (K(d) = 0.26 microM in 10 mM HEPES at pH 7.2 and 25 degrees C). However, at higher salt concentrations, the binding becomes much weaker, indicating the importance of electrostatic interactions. DHC is also very efficient in electron transfer to SHP with a second-order rate constant of 1.8 x 10(7) M(-)(1) s(-)(1) (at pH 7.2, 10 degrees C, and I = 500 mM). The reduction potentials of DHC and SHP are also suitably ordered for a favorable reaction with the hemes of DHC showing potentials of -310 and -240 mV, respectively, and that for SHP being -105 mV. These potentials are unaltered upon complex formation.     

Conferment of folding ability to a naturally unfolded apocytochrome c through introduction of hydrophobic amino acid residues

Hyperthermophilic Aquifex aeolicus cytochrome c(555) (AA c(555)) exceptionally folds even in the apo state, unlike general cytochromes c including mesophilic Pseudomonas aeruginosa cytochrome c(551) (PA c(551)), which is structurally homologous to AA c(555) in the holo state. Here we hypothesized that the exceptional apo AA c(555) folding can be attributed to nine hydrophobic amino acid residues and proved this using a PA c(551) variant (denoted as PA-nh) carrying the nine hydrophobic residues at structurally corresponding positions. Circular dichroism experiments showed that the apo PA-nh variant became folded, unlike the wild-type apo PA c(551), and exhibited much higher stability than the wild type. Another difference between the holo forms of AA c(555) and PA c(551) is the existence of an extra helix in the former. Introduction of the amino acid residues forming the extra helix of AA c(555) into the PA-nh variant did not significantly affect its folding ability in the apo state. Therefore, the nine hydrophobic residues introduced into the apo PA-nh variant were enough to confer the folding ability. PA c(551) represents the first example of the conversion of an intrinsically unfolded apocytochrome c into an autonomously folded one, which was revealed by means of a protein engineering method without heme. Although heme is generally considered to be a trigger of apocytochrome c folding, the present results demonstrate a new heme-independent folding mechanism.     

A comparison of the physical and chemical properties of four cytochromes c from Azotobacter vinelandii

1. A modified method for the separation and purification of four cytochromes c from Azotobacter vinelandii is described. Two new cytochromes c have been purified and are designated cytochromes c(551) and c(555). 2. Additional evidence is presented to establish the dihaem nature of cytochrome c(4). Ultracentrifugation data indicated similar molecular weights for the native and the denatured protein. Cleavage with CNBr yielded seven peptides; the amino acid compositions of the purified peptides were determined. Only one haem peptide was recovered. 3. Cytochromes c(551) and c(555) were characterized as acidic proteins of molecular weights about 12000. The spectral properties, isoelectric points, ;maps' of peptides from CNBr cleavage and amino acid compositions were determined for these two proteins. 4. The spectral properties, isoelectric points, molecular weights, CNBr peptide ;maps', amino acid compositions, relative oxidation-reduction potentials and e.p.r. (electron-paramagnetic-resonance) spectra of the four cytochromes c were compared. Cytochrome c(4) and cytochrome c(551) appear to be distinct proteins. The distinction between cytochromes c(5) and c(555) was not as clear, and our data are inadequate to establish firmly that they are distinct proteins. 5. The dihaem nature of cytochrome c(4) is evident in its e.p.r. spectrum. The e.p.r. spectra are similar to the spectra of mammalian cytochromes c.     

Studies on partially reduced mammalian cytochrome oxidase reactions with ferrocytochrome c.

The kinetics of the electron-transfer process which occurs between ferrocytochrome c and partially reduced mammalian cytochrome oxidase were studied by the rapid spectrophotometric techniques of stopped flow and temperature jump. Stopped-flow experiments showed initial very fast extinction changes at 605 nm and at 563 nm, indicating the simultaneous reduction of cytochrome a and oxidation of ferrocytochrome c. During this 'burst' phase, say the first 50 ms after mixing, it was invariably found that more cytochrome c had been oxidized than cytochrome a had been reduced. This discrepancy in electron equivalents may be accounted for by the rapid reduction of another redox site in the enzyme, possibly that associated with the extinction changes observed at 830 nm. During the incubation period in which the partially reduced oxidase was prepared, the rate of reduction of cytochrome a by ferrocytochrome c, at constant reactant concentrations, decreased with time. Temperature-jump experiments showed the presence of two relaxation processes. The faster of the two phases was assigned to the electron-transfer reaction between cytochrome c and cytochrome a. A study of the concentration-dependence of the reciprocal relaxation time for this phase yielded a rate constant of 9 X 10(6)M-1-s-1 for the electron transfer from cytochrome c to cytochrome a, and a value of 8.5 X 10(6)M-1-s-1 for the reverse reaction. The equilibrium constant for the electron-transfer reaction is therefore close to unity. The slower phase has been interpreted as signalling the transfer of electrons between cytochrome a and another redox site within the oxidase molecule.     

Three-dimensional structure of the nonaheme cytochrome c from Desulfovibrio desulfuricans Essex in the Fe(III) state at 1.89 A resolution

A nine heme group containing cytochrome c isolated from the soluble and membrane fractions of Desulfovibrio desulfuricans Essex, termed nonaheme cytochrome c, was crystallized, and the structure was solved using the multiple wavelength anomalous dispersion (MAD) phasing method. Refinement was carried out to a resolution of 1.89 A, and anisotropic temperature factors were addressed to the iron and sulfur atoms in the model. The structure revealed two cytochrome c(3) like domains with the typical arrangement of four heme centers. Both domains flanked an extra heme buried under the protein surface. This heme is held in position by loop extensions in each of the two domains. Although both the N- and C-terminal tetraheme domains exhibit a fold and heme arrangement very similar to that of cytochrome c(3), they differ considerably in their loop extensions and electrostatic surface. Analysis of the structure provides evidence for a different function of both domains, namely, anchoring the protein in a transmembranous complex with the N-terminal domain and formation of an electron-transfer complex with hydrogenase by the C-terminal domain.     

Unveiling a hidden folding intermediate in c-type cytochromes by protein engineering

Several investigators have highlighted a correlation between the basic features of the folding process of a protein and its topology, which dictates the folding pathway. Within this conceptual framework we proposed that different members of the cytochrome c (cyt c) family share the same folding mechanism, involving a consensus partially structured state. Pseudomonas aeruginosa cyt c(551) (Pa cyt c(551)) folds via an apparent two-state mechanism through a high energy intermediate. Here we present kinetic evidence demonstrating that it is possible to switch its folding mechanism from two to three state, stabilizing the high energy intermediate by rational mutagenesis. Characterization of the folding kinetics of one single-site mutant of the Pa cyt c(551) (Phe(7) to Ala) indeed reveals an additional refolding phase and a fast unfolding process which are explained by the accumulation of a partially folded species. Further kinetic analysis highlights the presence of two parallel processes both leading to the native state, suggesting that the above mentioned species is a non obligatory on-pathway intermediate. Determination of the crystallographic structure of F7A shows the presence of an extended internal cavity, which hosts three "bound" water molecules and a H-bond in the N-terminal helix, which is shorter than in the wild type protein. These two features allow us to propose a detailed structural interpretation for the stabilization of the native and especially the intermediate states induced by a single crucial mutation. These results show how protein engineering, x-ray crystallography and state-of-the-art kinetics concur to unveil a folding intermediate and the structural determinants of its stability.     

Reconstitution of the ubiquinol: cytochrome c reductase from a bc1 subcomplex and the 'Rieske' iron-sulfur protein isolated by a new method

1. A method for preparing the 'Rieske' iron-sulfur protein and the bc1 subcomplex of complex III was developed. The new method is advantageous over the published ones in that: (a) the final yield and amount exceeds by far those obtained when employing the hitherto published methods; (b) the iron-sulfur protein as well as the bc1 subcomplex are obtained by one and the same preparation procedure from a common source; and (c) the preparation method is easier than the published ones. 2. The iron-sulfur protein obtained represents the first reconstitutively active preparation present in a monodisperse state. 3. The reconstitution of the ubiquinol:cytochrome c reductase from the two components is a reversible dissociation process. Full activity of ubiquinol:cytochrome c reductase is reached after saturation of the binding site of the bc1 subcomplex for iron-sulfur protein. 4. Full reduction of the constituent cytochrome c1 of the bc1 subcomplex can already be obtained with substoichiometric amounts of iron-sulfur protein, however. 5. The question might be raised whether the observed dissociation equilibrium represents merely a phenomenon occurring specifically with the proteins isolated in Triton X-100 and investigated in a Triton-containing buffer, or whether dissociation of the iron-sulfur protein also takes place in the mitochondrial membrane in the course of the electron-transfer reaction sequence.     

Electron-transfer mechanism of the triplet state quenching of aluminium tetrasulfonated phthalocyanine by cytochrome c

The mechanism of electron-transfer from aluminium tetrasulfonated phthalocyanine triplet state to cytochrome c was investigated in this work. This reaction successfully quenches the dye triplet state due to the formation of complexes between the solute and the protein at the active site. The electron-transfer rate constant is around 3x10(7) s(-1), and is in accordance with previous results for the singlet excited state quenching [C.A.T. Laia, S.M.B. Costa, D. Phillips, A. Beeby. Electron-transfer kinetics in sulfonated aluminum phthalocyanines/cytochrome c complexes, J. Phys. Chem. B 108 (2004) 7506-7514.] in the framework of the Marcus theory, with a reorganization energy equal to 0.94 eV. The complex formation is diffusion controlled, but heterogeneities of the protein surface charge distribution lead to quenching rate constants smaller than predicted on a hard-spheres model with electrostatic interactions. Also the binding equilibrium constant is strongly affected by this phenomenon. Ionic strength plays an important role on the complex formation, but its effect on the unimolecular electron-transfer rate constant is negligible within experimental error.     

Use of surface enhanced infrared absorption spectroscopy (SEIRA) to probe the functionality of a protein monolayer

We present a novel infrared method to investigate the functionality of a protein monolayer tethered to a metal substrate. The approach employs Surface Enhanced Infrared Absorption Spectroscopy (SEIRAS), which renders high surface sensitivity by enhancing the signal of the adsorbed protein by up to approximately 2 orders of magnitude. We demonstrate that the electrochemically induced absorption changes of a cytochrome c monolayer can be observed with excellent signal-to-noise ratio when the protein is adhered to a modified gold surface. To probe membrane proteins, a concept is introduced for the oriented incorporation into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent is immobilized on a chemically modified gold surface via the affinity of its histidine (His)-tag to a nickel-chelating nitro-triacetic acid (NTA) surface. The protein monolayer is reconstituted into the lipid environment by detergent removal. Changing the orientation of the protein with respect to the metal surface is achieved by inserting the His-tag on either side of the membrane protein surface. Orientational control is mandatory for experiments in which electrons are injected from the electrode into the protein. The presented methodology opens new avenues to study the mechanism of the biomedically relevant class of electron and voltage-gated proteins on the atomic level.     

Peptide-protein interactions: photoinduced electron-transfer within the preformed and encounter complexes of a designed metallopeptide and cytochrome c

Photoinduced electron-transfer (ET) occurs between a negatively charged metallopeptide, [Ru(bpy)(2)(phen-am)-Cys-(Glu)(5)-Gly](3-) = RuCE(5)G, and ferricytochrome c = Cyt c. In the presence of Cyt c, the triplet state lifetime of the ruthenium metallopeptide is shortened, and the emission decays via biexponential kinetics, which indicates the existence of two excited-state populations of ruthenium peptides. The faster decay component displays concentration-independent kinetics demonstrating the presence of a preformed peptide-protein complex that undergoes intra-complex electron-transfer. Values of K(b) = (3.5 +/- 0.2) x 10(4) M(-1) and k(obs)(ET)= (2.7 +/- 0.4) x 10(6) s(-1) were observed at ambient temperatures. The magnitude of k(obs)(ET) decreases with increasing solvent viscosity, and the behavior can be fit to the expression k(obs)(ET) proportional to eta(-alpha) to give alpha = 0.59 +/- 0.05. The electron-transfer process occurring in the preformed complex is therefore gated by a rate-limiting configurational change of the complex. The slower decay component displays concentration-dependent kinetics that saturate at high concentrations of Cyt c. Analysis according to rapid equilibrium formation of an encounter complex that undergoes unimolecular electron-transfer yields K(b)' = (2.5 +/- 0.7) x 10(4) M(-1) and k(obs')(ET)= (7 +/- 3) x 10(5) s(-1). The different values of k(obs)(ET) and k(obs')(ET) suggest that the peptide lies farther from the heme when in the encounter complex. The value of k(obs')(ET) is viscosity dependent indicating that the reaction occurring within the encounter complex is also configurationally gated. A value of alpha = 0.98 +/- 0.14 is observed for k(obs')(ET), which suggests that the rate-limiting gating processes in the encounter complex is different from that in the preformed complex.     

Mechanism of ubiquinol oxidation by the bc(1) complex: role of the iron sulfur protein and its mobility

Native structures of ubihydroquinone:cytochrome c oxidoreductase (bc(1) complex) from different sources, and structures with inhibitors in place, show a 16-22 A displacement of the [2Fe-2S] cluster and the position of the C-terminal extrinsic domain of the iron sulfur protein. None of the structures shows a static configuration that would allow catalysis of all partial reactions of quinol oxidation. We have suggested that the different conformations reflect a movement of the subunit necessary for catalysis. The displacement from an interface with cytochrome c(1) in native crystals to an interface with cytochrome b is induced by stigmatellin or 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and involves ligand formation between His-161 of the [2Fe-2S] binding cluster and the inhibitor. The movement is a rotational displacement, so that the same conserved docking surface on the iron sulfur protein interacts with cytochrome c(1) and with cytochrome b. The mobile extrinsic domain retains essentially the same tertiary structure, and the anchoring N-terminal tail remains in the same position. The movement occurs through an extension of a helical segment in the short linking span. We report details of the protein structure for the two main configurations in the chicken heart mitochondrial complex and discuss insights into mechanism provided by the structures and by mutant strains in which the docking at the cytochrome b interface is impaired. The movement of the iron sulfur protein represents a novel mechanism of electron transfer, in which a tethered mobile head allows electron transfer through a distance without the entropic loss from free diffusion.     

Characterization of three soluble c-type cytochromes isolated from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705

Three soluble, low molecular mass cytochromes c (Mr 8000-15,000) were isolated and purified from soybean root nodule bacteroids of Bradyrhizobium japonicum strain CC705. On the basis of their alpha: absorbance peaks in the reduced forms, they were named cytochromes c550, c552 and c555. Cytochrome c552 reacted very fast, c555 very slowly and c550 not at all with carbon monoxide. The complete amino acid sequence (73 residues) of cytochrome c552 was established which identifies it as a monoheme, class I cytochrome c with some remote similarity to the cytochrome c6 family.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

TNF-alpha-induced apoptosis of macrophages following inhibition of NF-kappa B: a central role for disruption of mitochondria

Previously, we established that suppressing the constitutive activation of NF-kappaB in in vitro matured human macrophages resulted in apoptosis initiated by a decrease of the Bcl-2 family member, A1, and the loss of mitochondrial transmembrane potential (Deltapsi(m)). This study was performed to characterize the mechanism of TNF-alpha-induced apoptosis in macrophages following the inhibition of NF-kappaB. The addition of TNF-alpha markedly enhanced the loss of Deltapsi(m) and the induction of apoptotic cell death. Although caspase 8 was activated and contributed to DNA fragmentation, it was not necessary for the TNF-alpha-induced loss of Deltapsi(m). The inhibition of NF-kappaB alone resulted in the release of cytochrome c from the mitochondria, while both cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI were released following the addition of TNF-alpha. Furthermore, c-Jun N-terminal kinase activation, which was sustained following treatment with TNF-alpha when NF-kappaB was inhibited, contributed to DNA fragmentation. These observations demonstrate that cytochrome c and second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI may be differentially released from the mitochondria, and that the sustained activation of c-Jun N-terminal kinase modulated the DNA fragmentation independent of the loss of Deltapsi(m).