Yeast cytochrome c is a sequence-specific DNA-binding protein

A protein binding to the alcohol oxidase 2 upstream activation sequence (AOX2UAS) of the methylotropic yeast, Pichia pastoris, has been purified and identified as cytochrome c (cyt c). Cyt c purified from P. pastoris or Saccharomyces cerevisiae binds to AOX2UAS. Specific point mutations in AOX2UAS abolish cyt c binding. We conclude that yeast cyt c is a sequence-specific DNA-binding protein and may have a regulatory role in the nucleus.     

Understanding the mechanism of proton movement linked to oxygen reduction in cytochrome c oxidase: lessons from other proteins

Cytochrome c oxidase is a large intrinsic membrane protein designed to use the energy of electron transfer and oxygen reduction to pump protons across a membrane. The molecular mechanism of the energy conversion process is not understood. Other proteins with simpler, better resolved structures have been more completely defined and offer insight into possible mechanisms of proton transfer in cytochrome c oxidase. Important concepts that are illustrated by these model systems include the ideas of conformational change both close to and at a distance from the triggering event, and the formation of a transitory water-linked proton pathway during a catalytic cycle. Evidence for the applicability of these concepts to cytochrome c oxidase is discussed.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

A structural model for the adduct between cytochrome c and cytochrome c oxidase

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein–protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc ₁ to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc ₁–cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space. Protein Data Bank Accession numbers The coordinates of the five best structural models for each of the four clusters have been deposited in the Protein Data Bank (PDB ID 1ZYY).     

Stability and apoptotic activity of recombinant human cytochrome c

An efficient system for producing human cytochrome c variants is important to help us understand the roles of this protein in biological processes relevant to human diseases including apoptosis and oxidative stress. Here, we describe an Escherichia coli expression system for producing recombinant human cytochrome c. We also characterize the structure, stability, and function of the protein and show its utility for studying apoptosis. Yields of greater than 8 mg of pure protein per liter culture were attained. Circular dichroism spectropolarimetry studies show that the secondary and tertiary structures of the human protein are nearly identical to those of the horse protein, but the human protein is more stable than other eukaryotic cytochromes c. Furthermore, recombinant human cytochrome c is capable of inducing caspase-3 activity in a cell-free caspase activation assay. We use data from this assay along with data from the literature to define the apaf-1 binding site on human cytochrome c.     

Mitochondrial cytochrome c oxidase as a target site for cephalosporin antibiotics in renal epithelial cells (LLC-PK(1)) and renal cortex

We reported previously that treatment of the pig kidney proximal tubular epithelial cell line LLC-PK(1) with cephaloridine (CLD) decreased the activity of cytochrome c oxidase in the mitochondria of the cells followed by increases in lipid peroxidation and cell necrosis. In this study, we investigated the effects of CLD on the activity of cytochrome c oxidase in mitochondria isolated from LLC-PK(1) cells and purified the enzyme from mitochondria of the rat renal cortex. The activity of cytochrome c oxidase in the isolated mitochondria from LLC-PK(1) cells was significantly decreased from 1 h after addition of 1 mM CLD. Other cephalosporin antibiotics, cefazolin and cefalotin, also decreased the activity of cytochrome c oxidase in the isolated mitochondria. The activity of cytochrome c oxidase purified from the mitochondria of the rat renal cortex was also decreased from 2 h after addition of 1 mM CLD in a non-competitive manner. These results suggest that the direct inhibition of cytochrome c oxidase activity in the mitochondrial electron transport chain by cephlosporins may result from the observed nephrotoxicity.     

A novel 21-kDa cytochrome c-releasing factor is generated upon treatment of human leukemia U937 cells with geranylgeraniol

Geranylgeraniol (GGO) induces apoptosis in various lines of human tumor cells through a mitochondrion-dependent pathway. The present study describes identification of a 21-kDa cytochrome c-releasing factor that appears in the cytosolic fraction after treatment of human leukemia U937 cells with GGO. Incubation of isolated mitochondria with a lysate of U937 cells that had been treated with GGO resulted in the release of cytochrome c from the mitochondria. Utilizing this cell-free system, we purified a 21-kDa protein that induced the release of cytochrome c from mitochondria and appeared to be involved in the apoptosis that is induced in U937 cells by GGO. We designated this protein cytochrome c-releasing factor 21 (CRF21). Overexpression of CRF21 in HeLa cells induced the release of cytochrome c from mitochondria, with subsequent apoptosis. Our results suggest that CRF21 might play an important role in the induction of apoptosis by GGO in leukemia U937 cells.     

The orientations of cytochrome c in the highly dynamic complex with cytochrome b5 visualized by NMR and docking using HADDOCK

The interaction of bovine microsomal ferricytochrome b5 with yeast iso-1-ferri and ferrocytochrome c has been investigated using heteronuclear NMR techniques. Chemical-shift perturbations for 1H and 15N nuclei of both cytochromes, arising from the interactions with the unlabeled partner proteins, were used for mapping the interacting surfaces on both proteins. The similarity of the binding shifts observed for oxidized and reduced cytochrome c indicates that the complex formation is not influenced by the oxidation state of the cytochrome c. Protein-protein docking simulations have been performed for the binary cytochrome b5-cytochrome c and ternary (cytochrome b5)-(cytochrome c)2 complexes using a novel HADDOCK approach. The docking procedure, which makes use of the experimental data to drive the docking, identified a range of orientations assumed by the proteins in the complex. It is demonstrated that cytochrome c uses a confined surface patch for interaction with a much more extensive surface area of cytochrome b5. Taken together, the experimental data suggest the presence of a dynamic ensemble of conformations assumed by the proteins in the complex.     

A mechano-chemical model for energy transduction in cytochrome c oxidase: the work of a Maxwell's god

Cytochrome c3 has a central role in the energetics of Desulfovibrio sp., where it performs an electroprotonic energy transduction step. This process uses a network of cooperativities, largely based on anti-Coulomb components, resulting from a mechano-chemical energy coupling mechanism. This mechanism provides a model coherent with the data available for the redox chemistry of haem a of cytochrome c oxidase and its link to the activation of protons. A crucial feature of the model is an anti-Coulomb effect that sets the stage for a molecular ratchet, ensuring vectoriality for the redox-driven localised movement of protons across the membrane, against an electrochemical gradient.     

Galig, a novel cell death gene that encodes a mitochondrial protein promoting cytochrome c release

Galectin-3 internal gene (Galig) was recently identified as an internal gene transcribed from the second intron of the human galectin-3 gene that is implicated in cell growth, cell differentiation, and cancer development. In this study, we show that galig expression causes morphological alterations in human cells, such as cell shrinkage, cytoplasm vacuolization, nuclei condensation, and ultimately cell death. These alterations were associated with extramitochondrial release of cytochrome c, a known cell death effector. Furthermore, Bcl-xL co-transfection significantly reduced the release of cytochrome c induced by galig expression, suggesting a common pathway between the cytotoxic activity of galig and the anti-apoptotic activity of Bcl-xL. This antagonism was not observed upon co-transfection of Bcl-2 and galig. Galig encodes a mitochondrial-targeted protein named mitogaligin. Structure-activity relationship studies showed that the mitochondrial addressing of mitogaligin relies on an internal sequence that is required and sufficient for the release of cytochrome c and cell death upon cell transfection. Moreover, incubation of isolated mitochondria with peptides derived from mitogaligin induces cytochrome c release. Altogether, these results show that galig is a novel cell death gene encoding mitogaligin, a protein promoting cytochrome c release upon direct interaction with the mitochondria.     

Molecular mechanism of the inhibition of cytochrome c aggregation by Phe-Gly

Experimental and computational studies suggest that few general principles govern protein/protein interactions and aggregation. The knowledge of these rules may be exploited to design peptides that are able to interfere with the self-assembly and aggregation of proteins. This work is aimed to verify the validity of this hypothesis by investigating the interaction of cytochrome c with Phe and Gly amino acids, Ala-His (carnosine), and two water-soluble dipeptides Phe-Gly and Gly-Phe. The combined use of (1)H NMR, MD, and DSC has shown that: (i) at neutral pH, only Phe-Gly is able to prevent the thermally induced aggregation of cytochrome c; (ii) Phe-Gly interacts with Gly45 and Phe46 residues of the protein, either when the protein is in the folded or in the unfolded state; and (iii) the interaction of Phe-Gly with cytochrome c is sequence-dependent. These results support the hypothesis that the basic principles that describe protein aggregation can be used for the design of peptides with antiaggregating properties.     

A cytochrome c oxidase proton pumping mechanism that excludes the O2 reduction site

A redox-coupled conformational change in Asp51 of subunit I and a hydrogen-bond network connecting Asp51 with the matrix surface have been deduced from X-ray structures of bovine heart cytochrome c oxidase. This has provided evidence that Asp51 may play a role in the proton pumping process. However, the lack of complete conservation of a residue analogous to Asp51, the inclusion of a peptide bond in the hydrogen-bonding network and the lack of apparent involvement of the O2 reduction site have been used as arguments against the involvement of Asp51 in the mechanism of proton pumping. This minireview re-examines these arguments.     

Structure of a novel c7-type three-heme cytochrome domain from a multidomain cytochrome c polymer

The structure of a novel c(7)-type cytochrome domain that has two bishistidine coordinated hemes and one heme with histidine, methionine coordination (where the sixth ligand is a methionine residue) was determined at 1.7 A resolution. This domain is a representative of domains that form three polymers encoded by the Geobacter sulfurreducens genome. Two of these polymers consist of four and one protein of nine c(7)-type domains with a total of 12 and 27 hemes, respectively. Four individual domains (termed A, B, C, and D) from one such multiheme cytochrome c (ORF03300) were cloned and expressed in Escherichia coli. The domain C produced diffraction quality crystals from 2.4 M sodium malonate (pH 7). The structure was solved by MAD method and refined to an R-factor of 19.5% and R-free of 21.8%. Unlike the two c(7) molecules with known structures, one from G. sulfurreducens (PpcA) and one from Desulfuromonas acetoxidans where all three hemes are bishistidine coordinated, this domain contains a heme which is coordinated by a methionine and a histidine residue. As a result, the corresponding heme could have a higher potential than the other two hemes. The apparent midpoint reduction potential, E(app), of domain C is -105 mV, 50 mV higher than that of PpcA.     

Multi-step assembly pathway of the cbb3-type cytochrome c oxidase complex

The cbb3-type cytochrome c oxidases as members of the heme-copper oxidase superfamily are involved in microaerobic respiration in both pathogenic and non-pathogenic proteobacteria. The biogenesis of these multisubunit enzymes, encoded by the ccoNOQP operon, depends on the ccoGHIS gene products, which are proposed to be specifically required for co-factor insertion and maturation of cbb3-type cytochrome c oxidases. Here, the assembly of the cbb3-type cytochrome c oxidase from the facultative photosynthetic model organism Rhodobacter capsulatus was investigated using blue-native polyacrylamide gel electrophoresis. This process involves the formation of a stable but inactive 210 kDa sub-complex consisting of the subunits CcoNOQ and the assembly proteins CcoH and CcoS. By recruiting monomeric CcoP, this sub-complex is converted into an active 230 kDa CcoNOQP complex. Formation of these complexes and the stability of the monomeric CcoP are impaired drastically upon deletion of ccoGHIS. In a ccoI deletion strain, the 230 kDa complex was absent, although monomeric CcoP was still detectable. In contrast, neither of the complexes nor the monomeric CcoP was found in a ccoH deletion strain. In the absence of CcoS, the 230 kDa complex was assembled. However, it exhibited no enzymatic activity, suggesting that CcoS might be involved in a late step of biogenesis. Based on these data, we propose that CcoN, CcoO and CcoQ assemble first into an inactive 210 kDa sub-complex, which is stabilized via its interactions with CcoH and CcoS. Binding of CcoP, and probably subsequent dissociation of CcoH and CcoS, then generates the active 230 kDa complex. The insertion of the heme cofactors into the c-type cytochromes CcoP and CcoO precedes sub-complex formation, while the cofactor insertion into CcoN could occur either before or after the 210 kDa sub-complex formation during the assembly of the cbb3-type cytochrome c oxidase.     

Electron transfer among the CuA-, heme b- and a3-centers of Thermus thermophilus cytochrome ba3

The 1-methyl-nicotinamide radical (MNA(*)), produced by pulse radiolysis has previously been shown to reduce the Cu(A)-site of cytochromes aa(3), a process followed by intramolecular electron transfer (ET) to the heme a but not to the heme a(3) [Farver, O., Grell, E., Ludwig, B., Michel, H. and Pecht, I. (2006) Rates and equilibrium of CuA to heme a electron transfer in Paracoccus denitrificans cytochrome c oxidase. Biophys. J. 90, 2131-2137]. Investigating this process in the cytochrome ba(3) of Thermus thermophilus (Tt), we now show that MNA(*) also reduces Cu(A) with a subsequent ET to the heme b and then to heme a(3), with first-order rate constants 11200 s(-1), and 770 s(-1), respectively. The results provide clear evidence for ET among the three spectroscopically distinguishable centers and indicate that the binuclear a(3)-Cu(B) center can be reduced in molecules containing a single reduction equivalent.     

Activation of the cytochrome c peroxidase of Pseudomonas aeruginosa. The role of a heme-linked protein loop: a mutagenesis study

Mutagenesis studies have been used to investigate the role of a heme ligand containing protein loop (67-79) in the activation of di-heme peroxidases. Two mutant forms of the cytochrome c peroxidase of Pseudomonas aeruginosa have been produced. One mutant (loop mutant) is devoid of the protein loop and the other (H71G) contains a non-ligating Gly at the normal histidine ligand site. Spectroscopic data show that in both mutants the distal histidine ligand of the peroxidatic heme in the un-activated enzyme is lost or is exchangeable. The un-activated H71G and loop mutants show, respectively, 75% and 10% of turnover activity of the wild-type enzyme in the activated form, in the presence of hydrogen peroxide and the physiological electron donor cytochrome c(551). Both mutant proteins show the presence of constitutive reactivity with peroxide in the normally inactive, fully oxidised, form of the enzyme and produce a radical intermediate. The radical product of the constitutive peroxide reaction appears to be located at different sites in the two mutant proteins. These results show that the loss of the histidine ligand from the peroxidatic heme is, in itself, sufficient to produce peroxidatic activity by providing a peroxide binding site and that the formation of radical intermediates is very sensitive to changes in protein structure. Overall, these data are consistent with a major role for the protein loop 67-79 in the activation of di-heme peroxidases and suggest a "charge hopping" mechanism may be operative in the process of intra-molecular electron transfer.     

Characterization of the interaction of Rhodobacter capsulatus cytochrome c peroxidase with charge reversal mutants of cytochrome c(2)

Steady-state kinetics for the reaction of Rhodobacter capsulatus bacterial cytochrome c peroxidase (BCCP) with its substrate cytochrome c(2) were investigated. The Rb. capsulatus BCCP is dependent on calcium for activation as previously shown for the Pseudomonas aeruginosa BCCP and Paracoccus denitrificans enzymes. Furthermore, the activity shows a bell-shaped pH dependence with optimum at pH 7.0. Enzyme activity is greatest at low ionic strength and drops off steeply as ionic strength increases, resulting in an apparent interaction domain charge product of -13. All cytochromes c(2) show an asymmetric distribution of surface charge, with a concentration of 14 positive charges near the exposed heme edge of Rb. capsulatus c(2) which potentially may interact with approximately 6 negative charges, localized near the edge of the high-potential heme of the Rb. capsulatus BCCP. To test this proposal, we constructed charge reversal mutants of the 14 positively charged residues located on the front face of Rb. capsulatus cytochrome c(2) and examined their effect on steady-state kinetics with BCCP. Mutated residues in Rb. capsulatus cytochrome c(2) that showed the greatest effects on binding and enzyme activity are K12E, K14E, K54E, K84E, K93E, and K99E, which is consistent with the site of electron transfer being located at the heme edge. We conclude that a combination of long-range, nonspecific electrostatic interactions as well as localized salt bridges between, e.g., cytochrome c(2) K12, K14, K54, and K99 with BCCP D194, D241, and D6, account for the observed kinetics.     

hCOX18 and hCOX19: two human genes involved in cytochrome c oxidase assembly

We identified the human homologues of yCOX18 and yCOX19, two Saccharomyces cerevisiae genes involved in the biogenesis of mitochondrial respiratory chain complexes. In yeast, these two genes are required for the expression of cytochrome c oxidase: Cox18p catalyses the insertion of Cox2p COOH-tail into the mitochondrial inner membrane, and Cox19p is probably involved in metal transport to the intermembrane space. Both hCox18p and hCox19p present significant amino acid identity with the corresponding yeast polypeptides and reveal highly conserved functional domains. In addition, their subcellular localization is analogous to that of the yeast proteins. These data strongly suggest that the human gene products share similar functions with their yeast homologues. These two COX-assembly genes represent new candidates for mutational analysis in patients with isolated COX deficiency of unknown etiology.     

Overexpression of glutaredoxin 2 attenuates apoptosis by preventing cytochrome c release

Human mitochondrial glutaredoxin 2 (Grx2) catalyzes glutathione-dependent dithiol reaction mechanisms, reducing protein disulfides, and monothiol reactions, reducing mixed disulfides between proteins and GSH (de-/glutathionylation). Here, we have overexpressed Grx2 in HeLa cells in its mitochondrial form (mGrx2-HeLa) as well as a truncated cytosolic form, lacking the mitochondrial translocation signal (tGrx2-HeLa). The resulting clones were less susceptible to apoptosis induced by 2-deoxy-d-glucose (2-DG) or doxorubicin (Dox). Overexpression of Grx2 inhibited cytochrome c release and caspase activation induced by both agents. In addition, Grx2 prevented 2-DG- and Dox-induced loss of cardiolipin, the phospholipid anchoring cytochrome c to the inner mitochondrial membrane. Overexpression of mGrx2 provided better protection than tGrx2 overexpression, especially after treatment with 2-DG. We propose that Grx2 facilitates the maintenance of cellular redox homeostasis upon treatment with apoptotic agents, thereby preventing cardiolipin oxidation and cytochrome c release.     

Biogenesis of yeast mitochondrial cytochrome c: a unique relationship to the TOM machinery

The import of cytochrome c into the mitochondrial intermembrane space is not understood at a mechanistic level. While the precursor apocytochrome c can insert into protein-free lipid bilayers, the purified translocase of the outer membrane (TOM) complex supports the translocation of apocytochrome c into proteoliposomes. We report an in organello analysis of cytochrome c import into yeast mitochondria from wild-type cells and different mutants cells, each defective in one of the seven Tom proteins. The import of cytochrome c is not affected by removal of the receptor Tom20 or Tom70. Moreover, neither the transfer protein Tom5 nor the assembly factors Tom6 and Tom7 are needed for import of cytochrome c. When the general import pore (GIP)-protein Tom40 is blocked, the import of cytochrome c is moderately affected. Mitochondria lacking the central receptor and organizing protein Tom22 contain greatly reduced levels of cytochrome c. We conclude that up to two components of the TOM complex, Tom22 and possibly the GIP, are involved in the biogenesis of cytochrome c.