A study of the influence of the hydrophobic core residues of yeast iso-2-cytochrome c on phosphate binding: a probe of the hydrophobic core-surface charge interactions.

To gain insight into the role of hydrophobic core-surface charge interactions in stabilizing cytochrome c, we investigated the influence of hydrophobic core residues on phosphate binding by mutating residues in yeast iso-2-cytochrome c to those corresponding to iso-l-cytochrome c in various combinations. Heat transition of ultraviolet CD was followed as a function of pH in the presence and absence of phosphate. Thermodynamic parameters were deduced. It was found that the I20V/V43A/M98L mutation in the hydrophobic core, whose locations are remote from the putative phosphate sites, modulates phosphate interactions. The modulation is pH dependent. The I20V/ M98L and V43A mutation effects are nonadditive. The results lead to a model analogous to that of Tsao, Evans, and Wennerstrom, where a domain associated with the ordered hydrophobic core is sensitive to the fields generated by the surface charges. Such an explanation would be in accord with the observed difference in thermal stability between iso-2 and horse cytochromes c.

Functional correlation in amino acid residue mutations of yeast iso-2-cytochrome c that is consistent with the prediction of the concomitantly variable codon theory in cytochrome c evolution.

Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues I20, M64, L85, and M98 and surface residue L9 were mutated, in selected combinations, to those found in mammalian and bird cytochromes c. The functionality assay is based upon the ability of yeast cells to grow in YPGE medium. Furthermore, experiments on the single M64L and M98L mutations as well as the double M64L/M98L mutation using NMR showed that the effects of these mutations are to perturb the structural integrity of the protein. We identified functional correlation in two cases of a pair of residue mutations, the I20-->V and M98-->L pair and the L9-->I and L85-->I pair. In both cases, only one of the two alternative, putative evolutionary pathways leads to a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present data provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for the role of those specific atom-atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place in cytochrome c, in accordance with Fitch and Markowitz.

Antibody immunodiversity: a study on the marked specificity difference between two anti-yeast iso-1 cytochrome c monoclonal antibodies whose epitopes are closely related.

Anti-yeast iso-1 cytochrome c (cyt. c) monoclonal antibodies 2-96-12 and 4-74-6 have closely related epitopes (antigenic determinants). However, while the specificity of 4-74-6 is stringent, 2-96-12 cross-reacts with many evolutionarily related cytochromes c. Such a marked difference in specificity of antibodies with overlapping epitopes may represent unique antibody immunodiversity. Thus, we constructed Fv fragment models consisting of the variable domains of the heavy and light chains of 2-96-12 and 4-74-6 and that of another anti-iso-1 cyt. c as a control to gain insight into the origin of this difference in specificity. Our models show that 4-74-6 and 2-96-12 contain five and two aromatic side chains, respectively, in or near the central area of the antigen-combining site. The side chains of Arg95H (heavy chain) in 2-96-12 and Arg91L (light chain) in 4-74-6 project toward the central area of the combining site in our model. Antigen docking to our Fv models, combined with previous immunological studies, suggests that iso-1 cyt. c Asp60 may interact with Arg95H in 2-96-12 and Arg91L in 4-74-6 and that both epitopes of 2-96-12 and 4-74-7 may include iso-1 cyt. c Leu58, Asp60, Asn62, and Asn63. The effect of the Arg95H to Lys mutation on the antigen binding is also in accord with our model. The difference in specificity may be partly explained by a greater degree of conformational flexibility in and around the central area of the combining site in 2-96-12 compared to 4-74-6 due to differences in aromatic side chain packing.

[Cell to cell transfer of dense bodies induced by intracisternal infusion of leupeptin in rat cerebellum--quantitative and ultrastructural studies].

Previous studies have demonstrated that rats treated with a proteinase inhibitor, leupeptin (Leu), show an accumulation of lipofuscin (Lf)-like dense bodies in the neurons. The present study examines the quantitative and ultrastructural changes in the Lf-like dense bodies of Purkinje (P) cells and Bergmann (B) glia over 20 days following a 4-day regimen of Leu administration by intracisternal infusion. Toluidine blue staining revealed considerable Lf-like granules in both types of cells 1 day after Leu infusion. However, these granules had almost disappeared by 20 days after infusion. Quantitative analysis revealed a decrease in the amount of accumulated Lf-like granules in P cells whereas the granules in B glia increased 10 days after Leu infusion. Electron microscopic examination revealed the presence of Lf-like dense bodies on the plasma membranes of both the P cells and B glia. The present results suggest that Leu-induced dense bodies in P cells are transferred to B glia, indicating a possible route for the removal of Lf.

Intracellular metabolism of 99mTc-d,l-HMPAO in vitro: a basic approach for understanding the hyperfixation mechanism in damaged brain.

The mechanism of technetium-99m-labeled d,l-hexamethylpropylene amine oxime (99mTc-HMPAO) hyperfixation in damaged brain was elucidated using in vitro metabolic studies. Among the subcellular fractions of mouse brain homogenate, the mitochondrial fraction showed dominant metabolic activity with respect to 99mTc-HMPAO, followed by the cytosolic fraction. The metabolic activity of the mitochondrial fraction was enhanced by heat and detergent treatment, being proportional to the leakage of thiol (SH) compound(s) from the granules. The leaked SH compound(s) had a higher metabolic activity than glutathione, a well-known reductant in cells. 99mTc-HMPAO might be metabolized by mitochondrial SH compound(s) exhibiting strong reductant activity, and hyperfixation might be an indication of mitochondrial damage of the brain.

Hyperfixation of copper-62-PTSM in rat brain after transient global ischemia.

UNLABELLED: We evaluated the regional distribution of 62Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM), a potential PET perfusion agent, in the rat brain and observed hyperfixation in transient global ischemia in rats. METHODS: The distribution of 62Cu-PTSM was examined in comparison with that of 123I-labeled p-iodophenyl-N-isopropylmethanphetamine (123I-IMP) as a reference blood flow marker. Brain uptake of these two tracers was measured in Wistar rats subjected to 30-min four-vessel occlusion followed by recirculation for 10 min, 1 hr or 1, 3 or 5 days. Tracers were injected intravenously into rats 10 min before decapitation. The activities of Complex I and Complex I-III of mitochondria and the concentration of sulfhydryl (SH) groups were also measured. RESULTS: Copper-62-PTSM showed accelerated accumulation in the brain at 1 hr and 1 day after reperfusion when compared with that of 123I-IMP (p < 0.01), and this enhancement was considered to be due to hyperfixation. At these time points, SH concentration was significantly decreased (p < 0.01). On the other hand, the activity of Complex I was not influenced by ischemia/reperfusion, but that of Complex I-III was decreased to 65-70% of the control level (p < 0.01). CONCLUSIONS: Copper-62-PTSM showed hyperfixation most possibly as a result of increased NADH concentration, caused by disturbed electron transport in mitochondria.

Copper-62-ATSM: a new hypoxia imaging agent with high membrane permeability and low redox potential.

UNLABELLED: An ideal hypoxia imaging agent should have high membrane permeability for easy access to intracellular mitochondria and low redox potential to confer stability in normal tissue, but it should be able to be reduced by mitochondria with abnormally high electron concentrations in hypoxic cells. In this context, nitroimidazole residues are not considered to be essential. In this study, Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), a 62Cu-bisthiosemicarbazone complex, with high membrane permeability and low redox potential, was evaluated as a possible hypoxia imaging agent, using electron spin resonance spectrometry and the Langendorff isolated perfused rat heart model as well as rat heart left anterior descending occlusion model. METHODS: Nonradioactive Cu-ATSM was incubated with rat mitochondria, after which reduction of Cu(II) to Cu(I) was measured with electron spin resonance. As a model of hypoxic mitochondria, rotenone (Complex I inhibitor)-treated mitochondria were used. RESULTS: In this study, Cu-ATSM was reduced by hypoxic but not by normal mitochondria. CONCLUSION: Thus, retention of 62Cu-ATSM was studied serially in perfused rat hearts under conditions of normoxia (95% O2 + 5% CO2), hypoxia (95% N2 + 5% CO2) and reoxygenation (95% O2 + 5% CO2). In normoxia and reoxygenation, 62Cu-ATSM injected as a single bolus showed low retention (23.77% and 22.80%, respectively) 15 min after injection, but retention was increased markedly under hypoxic conditions (81.10%). Also, in the in vivo left anterior descending occluded rat heart model, 62Cu-ATSM retention was inversely correlated with accumulation of 201Tl, a relative myocardial blood flow marker.

Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM), a metal complex with selective NADH-dependent reduction by complex I in brain mitochondria: a potential radiopharmaceutical for mitochondria-functional imaging with positron emission tomography (PET).

The reductive retention mechanism of copper(II)-pyruvaldehyde-bis (N4-methylthiosemicarbazone) (Cu-PTSM), a generator-produced positron-emitting 62Cu-labeled radiopharmaceutical, was studied with non-radioactive and radioactive copper. Changes in the chemical form of Cu-PTSM were detected by electron spin resonance spectrometry (ESR) with cold copper. The effects of electron transport chain inhibitors on the reduction of Cu-PTSM were also examined. Rotenone and antimycin A activated the reduction of Cu-PTSM in the brain mitochondria by 1.6- and 1.4-fold, respectively, compared with untreated controls, while thenoyltrifluoroacetone (TTFA) had no effect on the reduction. These results were confirmed with radioactive copper. Furthermore, this reduction of Cu-PTSM was dependent on the protein concentration of mouse brain submitochondrial particle (SMP) with 1 mM NADH (0 mg-protein/ml: 1.8 +/- 2.5%, 8 mg-protein/ml: 69.0 +/- 5.5%, each value was % of reduced Cu). Similarly, this reduction depended on NADH concentration at a fixed concentration of SMP (8 mg-protein/ml). These results indicated that the electron transport chain, especially complex I, participated in the reduction of Cu-PTSM in brain mitochondria, and this suggested that Cu-PTSM has the potential to act as a functional imaging agent for diagnosis of the electron transport chain.

Differential mechanism of retention of Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) by brain and tumor: a novel radiopharmaceutical for positron emission tomography imaging.

The reductive retention of 62Cu-PTSM was comparatively studied in the brain and Ehrlich ascites tumor cells by electron spin resonance spectrometry and nonradioactive Cu-PTSM. In the brain, only the mitochondrial fraction showed the ability to reduce Cu-PTSM, and the other subcellular fractions did not. In contrast, the cytosolic fraction of Ehrlich ascites tumor cells was the specific site of Cu-PTSM reduction. It was therefore considered that the retention of Cu-PTSM in the brain is closely related to mitochondrial reduction, most probably involving the mitochondrial electron transport system.

Comparison of thermodynamic and kinetic effects between the Leu32-->norvaline and Leu35-->norvaline substitutions of the three-fragment complex of cytochrome c.

The three fragment complex (1-25)H.(28-38).(39-104) of horse cytochrome c (e.g., (1-25)H, the heme fragment containing residues 1 to 25) closely resembles the native protein except for residues 39 to 55, which are flexible. We have investigated how the Leu35-->Nva (norvaline) substitution differs from the Leu32-->Nva in the perturbation of the stability of this complex. The side chains of Leu32 and Leu35 are adjacent in the well-packed hydrophobic core of tuna cytochrome c (T. Takano and R. E. Dickerson (1981) J. Mol. Biol. 153, 95-115). We measured the effects of the substitutions on (i) the binding of (28-38) with ferri- and ferrocomplexes on the right side of the heme (Dickerson's orientation); (ii) the heat stability of the 695-nm band which monitors the Fe-S bond on the left side of the heme (cf. Takano and Dickerson, 1981); and (iii) the rate constant for the direct dissociation of (39-104) of the ferrous complex as a function of temperature. The results suggest that the Leu32-->Nva is unique in that the stabilizing energy associated with the ground state is markedly more perturbed in the Leu32-->Nva than in the Leu35-->Nva. This is true despite the fact that both substitutions introduce no stereochemical conflict and remove only the gamma-methyl groups of the Leu side chains which are presumably positioned adjacently with respect to each other in the core. Furthermore, the effect of the perturbation of the structure imposed by the removal of the Leu32 gamma-methyl group propagates itself perhaps through the core and affects the stability of the Fe-S bond and the binding strength of (39-104). These properties resemble the core domain-domain interaction (A. Fisher and H. Taniuchi (1992) Arch. Biochem. Biophys. 296, 1-16).

Effects of ischemia-reperfusion injury on myocardial single pass extraction and retention of Cu-PTSM in perfused rat hearts: comparison with 201T1 and 14C-iodoantipyrine.

The effects of ischemia-reperfusion-induced myocardial damage on the single pass extraction and retention of 64Cu-pyruvaldehyde-di(N4-methylthiosemicabazone) (64Cu-PTSM) in perfused rat hearts were compared to these effects on that of 201T1 and 14C-iodoantipyrine. 201T1 and 14C-iodoantipyrine did not show significant changes, but in the case of 64Cu-PTSM, the single pass extraction and retention was reduced with reperfusion. These findings indicate that ischemia-reperfusion-induced myocardial damage decrease the generator-produced 62Cu-labeled 62Cu-PTSM extraction and retention, and that 62Cu-PTSM might have potential not only as a blood flow tracer but also as a functional tracer.

Development of glycoside-bound radiopharmaceuticals: novel radioiodination method for digoxin.

We combined 2-hydroxy-3-methylbenzoylhydrazide (HMBH) with glycosides as a novel method for the radioiodination of physiologically active glycosides. This method was tested using digoxin, which is one of the cardiac glycosides. A digoxin-HMBH conjugate was synthesized by periodate cleavage of the third sugar ring, and was readily radiolabeled with Na[125I] by the chloramine-T method. 125I labelled digoxin-HMBH conjugate retained Na+, K(+)-ATPase binding in vivo and in vitro, and also retained immunoreactivity to an anti-digoxin antibody. Thus, this 125I labelled digoxin-HMBH conjugate represents a potential radiopharmaceutical for Na+, K(+)-ATPase imaging, as well as for the radioimmunoassay of digoxin.

Basic evaluation of 67Ga labeled digoxin derivative as a metal-labeled bifunctional radiopharmaceutical.

To develop metal-labeled digoxin radiopharmaceuticals with affinity with anti-digoxin antibody as well as Na+,K(+)-ATPase, a digoxin derivative conjugated with deferoxamine was synthesized. The derivative had a high binding affinity with 67Ga at deferoxamine introduced to the terminal sugar ring of digoxin. The 67Ga labeled digoxin derivative showed enough in vitro binding affinity and selectivity to anti-digoxin antibody as well as Na+,K(+)-ATPase. The 67Ga labeled digoxin derivative is considered to be a potential metal-labeled bifunctional radiopharmaceutical for digoxin RIA as well as myocardial Na+,K(+)-ATPase imaging.

Mitochondria-selective reduction of 62Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM) in the murine brain; a novel radiopharmaceutical for brain positron emission tomography (PET) imaging.

The retention mechanism of 62Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (62Cu-PTSM) in the murine brain was evaluated. For this purpose, stable Cu-PTSM was subjected to electron spin resonance spectrometry (ESR) and high performance liquid chromatography (HPLC) analysis to determine the valence state, coordination structure and tissue metabolism. In murine brain homogenate, ESR and HPLC analysis indicated the reduction and cleavage of Cu(II)-PTSM to Cu(I). This virtually irreversible reduction was specifically initiated by the mitochondrial enzymatic system in the murine brain.

A study of hydrogen exchange of monoclonal antibodies: specificity of the antigen-binding induced conformational stabilization.

Amide-hydrogen exchange of three anti-yeast iso-1-cytochrome-c IgG monoclonal antibodies and the Fab, prepared from one of them, were studied by infrared spectrophotometry in the presence and absence of the deuterated immunogen and evolutionarily related species (the deuterated immunogen contained a population of a dimer. Each subunit of the dimer appeared to bind to the antibodies in a manner similar to the monomer). The number of hydrogens of the antibodies whose exchange was suppressed on binding to the immunogen was found to exceed that estimated for the residues shielded by the immunogen. Analysis of the data suggests that such suppression of hydrogen exchange occurs mainly for the Fab domains, but not for the Fc. One of the antibodies showed two distinct classes of amide-hydrogens. Class-1 hydrogens (approx. 36/site) exchange faster than class 2 (approx. 37/site). The exchange of class-1 hydrogens was suppressed by binding to the immunogen, but not to the evolutionarily related species. The exchange of class-2 hydrogens was suppressed by binding to the evolutionarily related species, as well as to the immunogen. Thus, the suppression of exchange of class-1 hydrogens appears to occur by some kind of conformational stabilization, the mechanism of which differentiates between the deuterated immunogen and the evolutionarily related species. Evidence suggests that the trans-interactions of the Fab domains may modulate the hydrogen exchange. If it is assumed that the antigen-binding strengthens the trans-interactions in such a way that the exchange of the slower exchanging hydrogens is suppressed, this could explain the suppression of exchange of class-2 hydrogens.

A study of core domains, and the core domain-domain interaction of cytochrome c fragment complex.

To gain insight into the folding mechanism of the cytochrome c complex, we prepared a complete set of homologous and hybrid two-fragment ferric complexes of four different types and related complexes from horse, tuna, yeast iso-l, and Candida cytochromes c. The complexes were characterized for structural properties. Apparent equilibrium constants of the complexes were determined to calculate delta G0 for binding. The results have allowed us to assign four core domains of the complex. A core domain is a structural region containing a hydrophobic core and the surrounding shell which folds and unfolds as a unit. Core domain 1 folds by itself and consists essentially of the right channel structure, found by R. E. Dickerson and colleagues, and a part of the heme. Core domains 2, 3, and 4, respectively, are assigned based on the cores located on the left (the Fe-S bond) and right sides and at the bottom of heme. Evidence of the core domain-domain interaction to stabilize the Fe-S bond, combined with the kinetic studies by G. R. Parr and H. Taniuchi, has led to a model of two alternative folding orders of the core domains for the horse type I complex: domain 1----3----2----4 or 1----2----3----4. Furthermore, delta G0 variation between the complexes has shown non-additive behavior, indicating the existence of a residue-residue interaction between the heme- and apofragments in the complex. Evidence suggests that this interaction in most cases occurs within or through the core groups of the ordered interface between the heme- and the apo-fragments formed by folding of core domains 1, 2, and 3. Evidence also suggests that such core group interaction manifests itself in the interaction to stabilize the Fe-S bond and may be manifested in the core domain-domain interaction.

Complexation which facilitates rejoining of horse cytochrome c apofragment [Homoser-lactone65](1-65) or [Homoser-lactone65] (23-65) to apofragment (66-104).

We have shown that two CNBr fragments of horse apocytochrome c, [Homoser-lactone65](1-65) and (66-104), bind to the ferric heme fragment (1-25)H to form a non-productive three-fragment complex, and that when the heme of this complex has been kept reduced for 48 h at 25 degrees, the peptide bond between residues 65 and 66 is restored with a yield of 24% or more. We have also shown that another CNBr fragment [Homoser-lactone65](23-65), but not [Homoser-lactone65](39-65), similarly rejoins to fragment (66-104) in the presence of the ferrous heme fragment with 25% or more yield. For complex of ferro-heme fragment [Hse-lacton65](1-65)H and apofragment (66-104) of horse cytochrome c, which restores the peptide bond between residues 65 and 66 (located on the left side of the heme) (cf. Harbury, H.A. (1978) in Semisynthetic Peptides and Proteins (Offord, R.E. & DiBello, C., eds.), pp. 73-89, Academic Press, New York). Corradin & Harbury have suggested that axial ligation of methionine 80 to the heme (on the left side) is important. Consistent with their idea, fragment [Hse80](66-104) was found not to link to [Hse-lactone65](1-65) in the presence of ferro(1-25)H. Furthermore, the present studies indicate that the interaction involving residues 26 to 38 (on the right side) is also important for such a conformation which assists in the rejoining of the two apofragments. Combining these two ideas, we suggest that restoration of the peptide bond between residues 65 and 66 reflects the structural integrity of these complexes in the reduced form. Thus, the present reaction system can be used not only for chemical synthesis of [Homoser65] apocytochrome c but also to extend amino acid substitution studies of cytochrome c to residues 1 to 64.

A study of fine specificity of monoclonal antibodies to yeast iso-1-cytochrome c.

Seven monoclonal antibodies, prepared to yeast holo- or apo-iso-1-cytochrome c by the method of Köhler and Milstein (Goding, J. W. (1983) Monoclonal Antibodies: Principles and Practice, Academic Press, Orlando, FL) were characterized by cross-reaction with a panel of evolutionarily related cytochromes c, apocytochromes c, fragments and homologous and hybrid fragment complexes, inhibition, competitive inhibition, and complementation and fluorescence titration. The results have permitted us to assign the specifically recognized amino acids as follows. IgG1 monoclonals: 4-74-6, Leu-63 and/or Asn-67 and/or Asn-68; 4-128-6, Glu-93; 4-145-10, Thr-74; 2-96-12, Asp-65; 2-34-19, Lys-59; and 10-28-86, trimethyl-lysine 77. IgM monoclonal 39-14, Pro-30 and His-31. With mAb 4-128-6 substitution of glutamic acid 93 with alanine, as it occurs in Candida cytochrome c, has resulted in a decrease in affinity by a factor of 10(4). A calculation appears to show that this value is too large to be accounted for solely by the sum of energy losses due to disruption of charge neutralization and changes of hydrophobic interaction including van der Waals interaction. This and similar results with mAb 10-28-86 have led us to the idea that some new extra interatomic interaction sensitive to differences in configuration of atomic groups may be present and perturbed in the substitution. Furthermore, the assumption of the presence of such interaction can explain the striking similarity between the antigen-antibody interaction (e.g. Geysen, H. M., Meloen, R. H., and Barteling, S. J. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3998-4002) and a model system of horse cytochrome c three-fragment complex (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711) with respect to the high specificity of interacting residues in the interface. Thus, by analogy to the hypothesis developed in the model system (Fisher, A., and Taniuchi, H. (1988) FASEB J. 2, A1338), we hypothesize that a closed interaction loop would be formed on the basis of contacting groups including glutamic acid 93 across or within the interface between the antigen and mAb 4-128-6 and mediate delocalized interaction to generate extra binding energy.

Intramolecular flip between two alternative forms of complex formed from a heme fragment and apoprotein of horse cytochrome c.

The previous studies have shown that (a) noncovalent interactions of the ferro-heme fragment of residues 1-38 and apoprotein (1-104) of horse cytochrome c simultaneously and specifically form two isomeric complexes, types I and II resembling the native protein (the redundant residues flexibly protruding from the ordered structure); (b) the type II form but not type I appears to bind to CO; and (c) residues 39-55 are more flexible for type II form than type I (Parr, G. R., and Taniuchi, H. (1981) J. Biol. Chem. 256, 125-132). In the present study, we investigated 1) kinetics and thermodynamics of interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104); 2) the properties of the CO binding population; 3) the rate of dissociation of complexes ferri- and ferro-(1-38)-H.(39-104) (mimicking type II form); and 4) thermal transition of the 695-nm absorption band and biological activity of complexes. The results indicate (a) interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104) occurs without going through dissociation (t1/2 less than or equal to 12 min at 10 degrees C) and is associated with delta H (= -7.2 +/- 3.7 kcal/mol at 10 degrees C) favoring type I form and delta S (= 23 +/- 13 e.u. at 10 degrees C) favoring type II; (b) the CO-binding population correlates with type II; and (c) change from the ferrous to the ferric state of heme appears to perturb the thermodynamic relationship between type I and II forms. Interpreting the results and available evidence, we suggest that "intramolecular" flip between ferro-type I and ferro-type II forms would establish the Boltzmann distribution of these two distinctly different energy states, type I form having more strengthened interatomic interactions and type II more pronounced internal motion.

A study of roles of evolutionarily invariant proline 30 and glycine 34 of cytochrome c.

The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X (39-104) of horse cytochrome c, have shown that invariant leucine 32 and partially invariant leucine 35, both buried in the interior, exhibit a striking difference in perturbation of binding fragment (28-38) by substitution with isoleucine. Then the idea has been proposed that the energy states of leucine 32, the Met-80-S-heme-Fe bond and other distant residues such as tryptophan 59 would be coupled to generate extra force while leucine 35 would be less important for such coupling if it were involved. In the present studies we synthesized three (28-38) analogs substituting invariant proline 30 with glycine or invariant glycine 34 with alanine or serine. Thermodynamic and kinetic studies and UV CD and biological activity measurements were carried out on binding of the analogs to complex (1-25)H X (39-104). The results with the ferric form show that perturbations of delta G, delta H, and delta S associated with formation of the intermediate complex and with the ensuing process by the Gly34----Ala or Ser substitution result in weakening the Met-80-S-heme-Fe bond formed in the latter process; in contrast, perturbation by the Pro30----Gly substitution is small. However, the biological activity is more perturbed by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in the Gly34----Ala or Ser substitution the complex appears to be more readily activated for both formation and disruption of the Met-80-S-heme-Fe bond at 20 degrees C and below than without substitution. In all cases reduction of the heme strengthens the binding of fragment (28-38). However, striking are the increases in perturbation (less negative) of both delta H and delta S for binding of fragment (28-38) to form the ground state on reduction of the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies), and Leu32----norvaline (the previous studies) substitutions. It is known that fluctuation of the atomic positions of most residues of tuna ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation of the heme and that these three residues are among those showing the least fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26, Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT 400 WORDS)