Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

Cytochrome c at charged interfaces. 2. Complexes with negatively charged macromolecular systems studied by resonance Raman spectroscopy

We have analyzed the structure of cytochrome c (cyt c) bound in a variety of complexes in which negatively charged molecular groups interact with the positively charged binding domain around the heme crevice of cyt c. Using resonance Raman spectroscopy, we could demonstrate that these interactions induce the same conformational changes as they were observed in the surface-enhanced resonance Raman experiments of cyt c adsorbed on the Ag electrode [Hildebrandt & Stockburger (1989) Biochemistry (preceding paper in this issue)]. When cyt c is bound to (As4W40O140)27-, state II is stabilized, whereas in complexes with phosvitin and cytochrome b5 state I is formed. The complexes with phospholipid vesicles and inverted micelles reveal a mixture of both states. It is suggested that these systems as well as cyt c adsorbed on the Ag electrode may be regarded as model systems for the physiological complexes of cyt c with cytochrome oxidase and cytochrome reductase. On the basis of our findings it is proposed that the biological electron-transfer reactions are controlled by electric field induced conformational transitions of cyt c upon complex formation with its physiological redox partners.     

Effect of mutations in the cytochrome b ef loop on the electron-transfer reactions of the Rieske iron-sulfur protein in the cytochrome bc1 complex

Long-range movement of the Rieske iron-sulfur protein (ISP) between the cytochrome (cyt) b and cyt c1 redox centers plays a key role in electron transfer within the cyt bc1 complex. A series of 21 mutants in the cyt b ef loop of Rhodobacter sphaeroides cyt bc1 were prepared to examine the role of this loop in controlling the capture and release of the ISP from cyt b. Electron transfer in the cyt bc1 complex was studied using a ruthenium dimer to rapidly photo-oxidize cyt c1 within 1 mus and initiate the reaction. The rate constant for electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c1 was k1 = 60 000 s-1. Famoxadone binding to the Qo site decreases k1 to 5400 s-1, indicating that a conformational change on the surface of cyt b decreases the rate of release of the ISP from cyt b. The mutation I292A on the surface of the ISP-binding crater decreased k1 to 4400 s-1, while the addition of famoxadone further decreased it to 3000 s-1. The mutation L286A at the tip of the ef loop decreased k1 to 33 000 s-1, but famoxadone binding caused no further decrease, suggesting that this mutation blocked the conformational change induced by famoxadone. Studies of all of the mutants provide further evidence that the ef loop plays an important role in regulating the domain movement of the ISP to facilitate productive electron transfer and prevent short-circuit reactions.     

Characterization and calculation of a cytochrome c-cytochrome b5 complex using NMR data

To identify the binding site for bovine cytochrome b(5) (cyt b(5)) on horse cytochrome c (cyt c), cross-saturation transfer NMR experiments were performed with (2)H- and (15)N-enriched cyt c and unlabeled cyt b(5). In addition, chemical shift changes of the cyt c backbone amide and side chain methyl resonances were monitored as a function of cyt b(5) concentration. The chemical shift changes indicate that the complex is in fast exchange, and are consistent with a 1:1 stoichiometry. A K(a) of (4 +/- 3) x 10(5) M(-)(1) was obtained with a lower limit of 855 s(-)(1) for the dissociation rate of the complex. Mapping of the chemical shift variations and intensity changes upon cross-saturation NMR experiments in the complex reveals a single, contiguous interaction interface on cyt c. Using NMR data as constraints, a protein docking program was used to calculate two low-energy model complex clusters. Independent calculations of the effect of the cyt b(5) heme ring current-induced magnetic dipole on cyt c were used to discriminate between the different models. The interaction surface of horse cyt c in the current experimentally constrained model of the cyt c-cyt b(5) complex is similar but not identical to the interface predicted in yeast cyt c by Brownian dynamics and docking calculations. The occurrence of different amino acids at the protein-protein interface and the dissimilar assumptions employed in the calculations can largely account for the nonidentical interfaces.     

Two distinct, sequence-specific DNA-binding proteins interact independently with the major replication pause region of sea urchin mtDNA.

We have identified a second DNA-binding protein in sea urchin embryo mitochondria, which interacts with a binding site in the major replication pause region, at the junction of the genes for ATP synthase subunit 6 and cytochrome c oxidase subunit III (COIII). We provisionally designate this protein mtPBP2, to distinguish it from the previously characterized mitochondrial pause-region binding protein mtPBP1, whose properties and binding site are quite distinct. The high-affinity binding site for mtPBP2 lies at the 5' end of the COIII gene, and exhibits partial dyad symmetry, although modification interference analysis indicates that recognition is complex. Binding of mtPBP2 to this site induces a bend of approximately 45 degrees in the DNA. Southwestern blots show that mtPBP1 and 2 are both single polypeptides, of apparent molecular weights 25 kD and 18 kD respectively. In vitro, mtPBP1 and mtPBP2 bind independently to their high-affinity sites, which are separated by about 50 bp.     

A Brownian dynamics study of the interactions of the luminal domains of the cytochrome b6f complex with plastocyanin and cytochrome c6: the effects of the Rieske FeS protein on the interactions

The availability of the structures of the cytochrome b6f complex (cyt b6f), plastocyanin (PC), and cytochrome c6 (cyt c6) from Chlamydomonas reinhardtii allowed us, for the first time, to model electron transfer interactions between the luminal domains of this complex (including cyt f and the Rieske FeS protein) and its redox partners in the same species. We also generated a model structure in which the FeS center of the Rieske protein was positioned closer to the heme of cyt f than observed in the crystal structure and studied its interactions with both PC and cyt c6. Our data showed that the Rieske protein in both the original crystal structure and in our modeled structure of the cyt b6f complex did not physically interfere with binding position or orientation of PC or cyt c6 on cyt f. PC docked on cyt f with the same orientation in the presence or the absence of the Rieske protein, which matched well with the previously reported NMR structures of complexes between cyt f and PC. When the FeS center of the Rieske protein was moved close to the heme of cyt f, it even enhanced the interaction rates. Studies using a cyt f modified in the 184-191 loop showed that the cyt f structure is a more important factor in determining the rate of complex formations than is the presence or the absence of the Rieske protein or its position with respect to cyt f.     

Protein interactions leading to conformational changes monitored by limited proteolysis: apo form and fragments of horse cytochrome c

Proteolysis experiments have been used to monitor the conformational transitions from an unfolded to a folded state occurring when the apo form of horse cytochrome c (cyt c) binds the heme moiety or when two fragments of cyt c form a native-like 1:1 complex. Proteinase K was used as a proteolytic probe, in view of the fact that the broad substrate specificity of this protease allows digestion at many sites along a polypeptide chain. The rather unfolded apo form of cyt c binds heme with a concomitant conformational transition to a folded species characterized by an enhanced content of helical secondary structure. While the holoprotein is fully resistant to proteolytic digestion and the apoprotein is digested to small peptides, the noncovalent complex of the apoprotein and heme exhibits an intermediate resistance to proteolysis, in agreement with the fact that the more folded structure of the complex makes the protein substrate more resistant to proteolysis. The noncovalent native-like complex of the two fragments 1-56 and 57-104 of cyt c, covering the entire polypeptide chain of 104 residues of the protein, is rather resistant to proteolysis, while the individual fragments are easily digested. Fragment 57-104 is fast degraded to several peptides, while fragment 1-56 is slowly degraded stepwise from its C-terminal end, leading initially mostly to fragments 1-48 and 1-40 and, at later stages of proteolysis, fragments 1-38, 1-35, 1-33, and 1-31. Thus, proteolysis data indicate that the heme containing fragment 1-56 has a rather compact core and a C-terminal flexible tail. Upon prolonged incubation of the complex of fragments 1-56 and 57-104 (nicked cyt c) with proteinase K, a chain segment is removed from the nicked protein, leading to a gapped protein complex of fragments of 1-48 and 57-104 and, on further digestion, fragments 1-40 and 57-104. Of interest, the chain segment being removed by proteolysis of the complex matches the omega-loop which is evolutionarily removed in cyt c of microbial origin. Overall, rates and/or resistance to proteolysis correlates well with the extent of folding of the protein substrates, as deduced from circular dichroism measurements. Thus, our results underscore the utility of proteolytic probes for analyzing conformational and dynamic features of proteins. Finally, a specific interest of the cyt c fragment system herewith investigated resides in the fact that the fragments are exactly the exon products of the cyt c gene.     

Multiple Q-cycle bypass reactions at the Qo site of the cytochrome bc1 complex

The cytochrome (cyt) bc(1) complex is central to energy transduction in many species. Most investigators now accept a modified Q-cycle as the catalytic mechanism of this enzyme. Several thermodynamically favorable side reactions must be minimized for efficient functioning of the Q-cycle. Among these, reduction of oxygen by the Q(o) site semiquinone to produce superoxide is of special pathobiological interest. These superoxide-producing bypass reactions are most notably observed as the antimycin A- or myxothiazol-resistant reduction of cyt c. In this work, we demonstrate that these inhibitor-resistant cyt c reductase activities are largely unaffected by removal of O(2) in the isolated yeast cyt bc(1) complex. Further, increasing O(2) tension 5-fold stimulated the antimycin A-resistant reduction by a small amount ( approximately 25%), while leaving the myxothiazol-resistant reduction unchanged. This most likely indicates that the rate-limiting step in superoxide production is the formation of a reactive species (probably a semiquinone), capable of rapid O(2) reduction, and that in the absence of O(2) this species can reduce cyt c by some other pathway. We suggest as one possibility that a semiquinone escapes from the Q(o) site and reduces either O(2) or cyt c directly. The small increase in antimycin A-resistant cyt c reduction rate at high O(2) can be explained by the accumulation of a low concentration of a semiquinone inside the Q(o) site. Under aerobic conditions, addition of saturating levels of superoxide dismutase (SOD) inhibited 50% of cyt c reduction in the presence of myxothiazol, implying that essentially all bypass reactions occur with the production of superoxide. However, SOD inhibited only 35% of antimycin A-resistant cyt c reduction, suggesting the presence of a second, slower bypass reaction that does not reduce O(2). Given that myxothiazol blocks cyt b reduction whereas antimycin A promotes it, we propose that this second bypass occurs by reduction of the Q(o) site semiquinone by prereduced cyt b(L).     

Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

Identification of precise electrostatic recognition sites between cytochrome c6 and the photosystem I subunit PsaF using mass spectrometry

The reduction of the photo-oxidized special chlorophyll pair P700 of photosystem I (PSI) in the photosynthetic electron transport chain of eukaryotic organisms is facilitated by the soluble copper-containing protein plastocyanin (pc). In the absence of copper, pc is functionally replaced by the heme-containing protein cytochrome c6 (cyt c6) in the green alga Chlamydomonas reinhardtii. Binding and electron transfer between both donors and PSI follows a two-step mechanism that depends on electrostatic and hydrophobic recognition between the partners. Although the electrostatic and hydrophobic recognition sites on pc and PSI are well known, the precise electrostatic recognition site on cyt c6 is unknown. To specify the interaction sites on a molecular level, we cross-linked cyt c6 and PSI using a zero-length cross-linker and obtained a cross-linked complex competent in fast and efficient electron transfer. As shown previously, cyt c6 cross-links specifically with the PsaF subunit of PSI. Mass spectrometric analysis of tryptic peptides from the cross-linked product revealed specific interaction sites between residues Lys27 of PsaF and Glu69 of cyt c6 and between Lys23 of PsaF and Glu69/Glu70 of cyt c6. Using these new data, we present a molecular model of the intermolecular electron transfer complex between eukaryotic cyt c6 and PSI.     

Binding of arylazidocytochrome c derivatives to beef heart cytochrome c oxidase: cross-linking in the high- and low-affinity binding sites

Two arylazidocytochrome c derivatives, one modified at lysine-13 and the second modified at lysine-22, were reacted with beef heart cytochrome c oxidase. The lysine-13 modified arylazidocytochrome c was found to cross-link both to the enzyme and with lipid bound to the cytochrome c oxidase complex. The lysine-22 derivative reacted only with lipids. Cross-linking to protein was through subunit II of the cytochrome c oxidase complex, as first reported by Bisson et al. [Bisson, R., Azzi, A., Gutweniger, H., Colonna, R., Monteccuco, C., & Zanotti, A. (1978) J. Biol. Chem. 253, 1874]. Binding studies show that the cytochrome c derivative covalently bound to subunit II was in the high-affinity binding site for the substrate. Evidence is also presented to suggest that cytochrome c bound to the lipid was in the low-affinity binding site [as defined by Ferguson-Miller et al. [Ferguson-Miller, S., Brautigan, D. L., & Margoliash, E. (1976) J. Biol. Chem. 251, 1104]]. Covalent binding of the cytochrome c derivative into the high-affinity binding site was found to inhibit electron transfer even when native cytochrome c was added as a substrate. Inhibition was almost complete when 1 mol of the Lys-13 modified arylazidocytochrome c was covalently bound to the enzyme per cytochrome c oxidase dimer (i.e., congruent to 280 000 daltons). Covalent binding of either derivative with lipid (low-affinity site) had very little effect on the overall electron transfer activity of cytochrome c oxidase. These results are discussed in terms of current theories of cytochrome c-cytochrome c oxidase interactions.     

Surface plasmon resonance studies of complex formation between cytochrome c and bovine cytochrome c oxidase incorporated into a supported planar lipid bilayer. II. Binding of cytochrome c to oxidase-containing cardiolipin/phosphatidylcholine membranes.

Complex formation between horse heart cytochrome c (cyt c) and bovine cytochrome c oxidase (cco) incorporated into a supported planar egg phosphatidylcholine membrane containing varying amounts of cardiolipin (CL) (0-20 mol%) has been studied under low (10 mM) and medium (160 mM) ionic strength conditions by surface plasmon resonance (SPR) spectroscopy. Both specific and nonspecific modes of cyt c binding are observed. The dissociation constant of the specific interaction between cyt c and cco increases from approximately 6.5 microM at low ionic strength to 18 microM at medium ionic strength, whereas the final saturation level of bound protein is independent of salt concentration and corresponds to approximately 53% of the total cco molecules present in the membrane. This suggests a 1:1 binding stoichiometry between the two proteins. The nonspecific binding component is governed by electrostatic interactions between cyt c and the membrane lipids and results in a partially ionic strength-reversible protein-membrane association. Thus, hydrophobic interactions between cyt c and the membrane, which are the predominant mode of binding in the absence of cco, are greatly suppressed. Both the amount of nonspecifically bound protein and the binding affinity can be varied over a broad range by changing the ionic strength and the extent of CL incorporation into the membrane. Under conditions approximating the physiological state in the mitochondrion (i.e., 20 mol% CL and medium ionic strength), 1-1.5 cyt c molecules are bound to the lipid phase per molecule of cco, with a dissociation constant of 0.1 microM. The possible physiological significance of these observations is discussed.     

A conformational change in cytochrome c of apoptotic and necrotic cells is detected by monoclonal antibody binding and mimicked by association of the native antigen with synthetic phospholipid vesicles

By flow cytometry, a conformational change in mouse cytochrome c (cyt c) of apoptotic and necrotic T hybridoma cells was detected using a monoclonal antibody (mAb) that recognizes the region around amino acid residue 44 on a non-native form of the protein. The conformational change in cyt c is an early event in apoptosis, which can be identified in pre-apoptotic cells that are negative for other indicators of apoptosis. Since the mAb did not bind fixed and permeabilized live cells and did not immunoprecipitate soluble cyt c extracted with detergent from dead cells, it appears to recognize cyt cbound in a detergent-sensitive complex to other cellular components. Coincidentally, the mAb was also shown by competitive enzyme-linked immunosorbent assay to bind cyt c associated with synthetic phosphatidic acid vesicles. This suggests that the conformational change of cyt c in dying cells could be due to its association with intracellular membranes that are, perhaps, altered in cell death. By immunofluorescent confocal microscopy, conformationally altered cyt c in post-apoptotic T hybridoma cells showed a punctate distribution, indicating that it remained associated with mitochondria. Furthermore, the heavy membrane fraction of post-apoptotic cells but not of live cells was functional in caspase activation. This suggests that membrane-bound cyt c is the relevant caspase coactivation factor in the T hybridoma cells.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Expression and characterization of the diheme cytochrome c subunit of the cytochrome bc complex in Heliobacterium modesticaldum

Heliobacterium modesticaldum is a Gram-positive, anaerobic, anoxygenic photoheterotrophic bacterium. Its cytochrome bc complex (Rieske/cyt b complex) has some similarities to cytochrome b(6)f complexes from cyanobacteria and chloroplasts, and also shares some characteristics of typical bacterial cytochrome bc(1) complexes. One of the unique factors of the heliobacterial cytochrome bc complex is the presence of a diheme cytochrome c instead of the monoheme cytochrome f in the cytochrome b(6)f complex or the monoheme cytochrome c(1) in the bc(1) complex. To understand the structure and function of this diheme cytochrome c protein, we expressed the N-terminal transmembrane-helix-truncated soluble H. modesticaldum diheme cytochrome c in Escherichia coli. This 25kDa recombinant protein possesses two c-type hemes, confirmed by mass spectrometry and a variety of biochemical techniques. Sequence analysis of the H. modesticaldum diheme cytochrome c indicates that it may have originated from gene duplication and subsequent gene fusion, as in cytochrome c(4) proteins. The recombinant protein exhibits a single redox midpoint potential of +71mV versus NHE, which indicates that the two hemes have very similar protein environments.     

Direct evidence for the cooperative unfolding of cytochrome c in lipid membranes from H-(2)H exchange kinetics

The interaction of cytochrome c (cyt c) with anionic lipid membranes is known to disrupt the tightly packed native structure of the protein. This process leads to a lipid-inserted denatured state, which retains a native-like alpha-helical structure but lacks any specific tertiary interactions. The structural and dynamic properties of cyt c bound to vesicles containing an anionic phospholipid (DOPS) were investigated by amide H-(2)H exchange using two-dimensional NMR spectroscopy and electrospray ionisation mass spectrometry. The H-(2)H exchange kinetics of the core amide protons in cyt c, which in the native protein undergo exchange via an uncorrelated EX2 mechanism, exchange in the lipid vesicles via a highly concerted global transition that exposes these protected amide groups to solvent. The lack of pH dependence and the observation of distinct populations of deuterated and protonated species by mass spectrometry confirms that exchange occurs via an EX1 mechanism with a common rate of 1(+/-0.5) h(-1), which reflects the rate of transition from the lipid-inserted state, H(l), to an unprotected conformation, D(i), associated with the lipid interface.     

Effect of polyanion on the acidic conformational transition of native and denatured ferricytochrome c. Circular dichroism study

Interaction of polyanion poly(vinylsulfate) with oxidized cytochrome c (cyt c) significantly affects the protein main characteristics. One of them, pKa value of acidic transition, was shifted from an apparent pKa value 2.5 (typical for cyt c in low ionic strength solvent) to approximately 5.20 +/- 0.15 upon polyanion binding to the protein, pointing to a likely involvement of histidines 26 and/or 33 in the protein acidic transition in complex with the polyanion. The acidic transition followed at 6 different wavelengths all over circular dichroism spectrum, monitoring different parts of the protein structure, revealed basically two-state character process. Only ellipticity at 262 nm indicated a low-cooperative pH-induced conformational transition in heme region with an apparent pKa approximately 4.34 +/- 0.25 in accordance with absorbance change at 620 nm. Polyanion also interacts with chemically-denatured (in the presence of 9 mol/l urea) state of the protein as it follows from stabilization of protein residual structure at acidic pH and its effect on pKa value of acidic transition of chemically-denatured cyt c. Destabilization effect of polyanions on native and, on the other hand, stabilization influence on partially unfolded conformations of the protein are discussed with an implication for their chaperone-like properties in vivo and in vitro.     

Thermal inactivation of electron-transport functions and F0F1-ATPase activities

Bovine heart submitochondrial particles in suspension were heated at a designated temperature for 3 min, then cooled for biochemical assays at 30 degrees C. By enzyme activity measurements and polarographic assay of oxygen consumption, it is shown that the thermal denaturation of the respiratory chain takes place in at least four stages and each stage is irreversible. The first stage occurs at 51.0 +/- 1.0 degrees C, with the inactivation of NADH-linked respiration, ATP-driven reverse electron transport, F0F1 catalyzed ATP/Pi exchange, NADH and succinate-driven ATP synthesis. The second stage occurs at 56.0 +/- 1.0 degrees C, with the inactivation of succinate-linked proton pumping and respiration. The third stage occurs at 59.0 +/- 1.0 degrees C, with the inactivation of electron transfer from cytochrome c to cytochrome oxidase and ATP-dependent proton pumping. The ATP hydrolysis activity of F0F1 persists to 61.0 +/- 1.0 degrees C. An additional transition, detectable by differential scanning calorimetry, occurring around 70.0 +/- 2.0 degrees C, is probably associated with thermal denaturation of cytochrome c and other stable membrane proteins. In the presence of either mitochondrial matrix fluid or 2 mM mercaptoethanol, all five stages give rise to endothermic effects, with the absorption of approx. 25 J/g protein. Under aerobic conditions, however, the first four transitions become strongly exothermic, and release a total of approx. 105 J/g protein. Solubilized and reconstituted F0F1 vesicles also exhibit different inactivation temperatures for the ATP/Pi exchange, proton pumping and ATP hydrolysis activities. The first two activities are abolished at 49.0 +/- 1.0 degrees C, but the latter at 58.0 +/- 2.0 degrees C. Differential scanning calorimetry also detects biphasic transitions of F0F1, with similar temperatures of denaturation (49.0 and 54.0 degrees C). From these and other results presented in this communication, the following is concluded. (1) A selective inactivation, by the temperature treatment, of various functions of the electron-transport chain and of the F0F1 complex can be done. (2) The ATP synthesis activity of the F0F1 complex involves either a catalytic or a regulation subunit(s) which is not essential for ATP hydrolysis and the proton translocation. This subunit is 10 degrees C less stable than the hydrolytic site. Micromolar ADP stabilizes it from thermal denaturation by 4-5 degrees C, although ADP up to millimolar concentration does not protect the hydrolytic site and the proton-translocation site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionic strength effects on cytochrome aa3 kinetics

1. The occurrence of an optimal ionic strength for the steady-state activity of isolated cytochrome aa3 can be attributed to two opposite effects: upon lowering of the ionic strength the affinity between cytochrome c and cytochrome aa3 increases, whereas in the lower ionic strength region the formation of a less active cytochrome c-aa3 complex limits the ferrocytochrome c association to the low affinity site. 2. At low ionic strength, the reduction of cytochrome c-aa3 complex by ferrocytochrome c1 proceeds via non-complex-bound cytochrome c. Under these conditions the positively charged cytochrome c provides the electron transfer between the negatively charged cytochromes c1 and aa3. 3. Polylysine is found to stimulate the release of tightly bound cytochrome c from the cytochrome c-aa3 complex. This property points to the existence of negative cooperativity between the two binding sites. We suggest that the stimulation is not restricted to polylysine, but also occurs with cytochrome c. 4. Dissociation rates of both high and low affinity sites on cytochrome aa3 were determined indirectly. The dissociation constants, calculated on the basis of pre-steady-state reaction rates at an ionic strength of 8.8 mM, were estimated to be 0.6 nM and 20 microM for the high and low affinity site, respectively.