Alkaline Isomerization Research Articles

Thermodynamic and kinetic studies of cytochrome c from different species

The direct heterogeneous electron transfer reactions of cytochrome c from different vertebrate species are compared in this report. A kinetic study revealed a biphasic temperature dependence at neutral pH for the formal heterogeneous electron transfer rate constants of these cytochromes. The properties of the electrode-solution interface are believed to cause this biphasic kinetic effect. This effect does not correlate with the conformational transitions evident in both formal potential and circular dichroism (CD) measurements as a function of temperature. The conformational transitions seen in the formal potential and CD measurements arise from the alkaline isomerization and heme crevice changes. These are subtle effects compared with the structural changes associated with unfolding of the protein that are evident in differential scanning calorimetric experiments. The conformational stabilities of cytochrome c between species relate to the differences in their amino acid sequences. The amino acid interactions which stabilize the structure of these different cytochrome c molecules have been evaluated and will be discussed.

The role of tyrosine 67 in the cytochrome c heme crevice structure studied by semisynthesis.

Tyr-67 of mitochondrial cytochrome c is thought to be involved in important hydrogen bonding interactions in the hydrophobic heme pocket of the protein (Takano, T., Dickerson, R. E. (1981) J. Mol. Biol. 153:95-115). The role of this highly conserved residue in heme pocket stability was studied by comparing properties of semisynthetic (Phe-67) and (p-F-Phe-67) analogs with those of native cytochrome c and a "control" analog, (Hse-65)cytochrome c. The (Phe-67) and (p-F-Phe-67) analogs have well-developed 695-nm visible absorption bands and are active in a cytochrome c oxidase assay. The reduction potentials of both analogs are lower than the native protein by approximately 50 mV. Although both analogs bind imidazole with higher affinity than the native protein, only the (p-F-Phe-67) analog has a 3- to 5-fold lower binding constant for cyanide. Only the (Phe-67) analog was significantly more stable toward alkaline isomerization. These results are not consistent with stabilization of the native protein heme pocket via hydrogen bonding of Tyr-67 to Met-80. An alternative steric role for Tyr-67 is proposed in which the residue controls the heme reduction potential by limiting the number of internal H2O molecules in the heme pocket.

Determination of naproxen and its metabolite O-desmethylnaproxen with their acyl glucuronides in human plasma and urine by means of direct gradient high-performance liquid chromatography

Naproxen is metabolized in humans by O-demethylation, and by acyl glucuronidation to the 1-O-glucuronide. Naproxen, its metabolite and the conjugates can be measured directly by gradient high-performance liquid chromatographic analysis without enzymic deglucuronidation. The glucuronide conjugates were isolated by preparative chromatography from human urine samples. Mild acidic hydrolysis of one urinary conjugate resulted in naproxen. This conjugate was also formed by alkaline isomerization of isolated naproxen acyl glucuronide, indicating that the structure of this urinary conjugate must have been naproxen isoglucuronide (4-O-glucuronide). Mild acidic hydrolysis of another urinary conjugate resulted in O-desmethylnaproxen. This conjugate was also formed by alkaline isomerisation of isolated O-desmethylnaproxen acyl glucuronide, indicating that the structure of this urinary conjugate must have been O-desmethylnaproxen isoglucuronide (4-O-glucuronide). Calibration curves were constructed by enzymic deconjugation of samples containing different concentrations of isolated naproxen acyl glucuronide, O-desmethylnaproxen acyl glucuronide, and the isoglucuronides of naproxen and O-desmethylnaproxen by mild acidic hydrolysis. The limit of quantitation of naproxen in plasma is 1.5 μg/ml. The limits of quantitation in urine are: naproxen, O-desmethylnaproxen, naproxen acyl glucuronide and O-desmethylnaproxen acyl glucuronide, 1 μg/ml; the isoglucuronide of naproxen and O-desmethylnaproxen, 1.5 μg/ml. A pharmacokinetic profile of naproxen is shown, and some preliminary pharmacokinetic parameters of naproxen obtained from two human volunteers are given.

1H-n.m.r. evaluation of the ferricytochrome c-cardiolipin interaction. Effect of superoxide radicals

The interaction between ferricytochrome c and cardiolipin was investigated by 1H n.m.r. at 270 MHz. From the phospholipid-induced changes of the protein spectral features it is concluded that the first 2 equivalents of cardiolipin cause a conformational change at the lower part of the solvent-exposed haem edge, involving a rearrangement of the hydrogen-bond interactions of propionate 6, thus partly accounting for the lowered redox potential of cytochrome c in the presence of cardiolipin. The increased value for the pK of the alkaline isomerization of ferricytochrome c shows that cardiolipin stabilizes the native structure of the protein, indicating that the oxidized form assumes ferrocytochrome c-like properties. Peroxidation of cardiolipin by superoxide radical ions drastically decreases the protein binding to this phospholipid. The implications of this finding, and the likelihood of the ternary cytochrome c-cardiolipin-cytochrome c oxidase complex, for the binding of cytochrome c to cytochrome c oxidase in vivo, are discussed in relation to peroxidative damage following ischaemia and reperfusion.

Mutation-induced perturbation of the cytochrome c alkaline transition

The possible influence of residue Phe-82 in the cytochrome c alkaline isomerization has been evaluated by spectrophotometric pH titrations of a family of mutant yeast iso-1-cytochromes c in which the identity of the residue at this position has been varied. The pKa for the exchange of the Met-80 heme iron ligand was determined from pH titrations in which the S----Fe charge-transfer band (695 nm) was monitored and was found to be 8.5 for the wild type, 7.7 for Ser-82, 7.7 for Gly-82, 7.2 for Leu-82, and 7.2 for Ile-82. pH-jump experiments [Davis et al. (1974) J. Biol. Chem. 249, 2624] established that substitutions at position 82 affect the alkaline isomerization by lowering the pKa of the titrating group by as much as 1.4 pK units; for the Ser-82 and Gly-82 variants, there is also a small effect on the Keq for the ligand exchange equilibrium. On the basis of these findings, we conclude that one critical role for Phe-82 in the wild-type protein is stabilization of the native heme binding environment.

NMR study of the alkaline isomerization of ferricytochrome c

The pH-induced isomerization of horse heart cytochrome c has been studied by 1H NMR. We find that the transition occurring in D 2O with a p K a measured as 9.5±0.1 is from the native species to a mixture of two basic forms which have very similar NMR spectra. The heme methyl peaks of these two forms have been assigned by 2D exchange NMR. The forward rate constant (native to alkaline cytochrome c) has a value of 4.0±0.6 s −1 at 27°C and is independent of pH; the reverse rate constant is pH-dependent. The activation parameters are Δ H ‡ = 12.8±0.8 kcal·mol 1, Δ S ‡ = − 12.9±2.0 e.u. for the forward reaction and Δ H ‡ = 6.0±0.3 kcal·mol −1, Δ S ‡ = − 35.1±1.3 e.u. for the reverse reaction (pH*=9.28). Δ H° and Δ S° for the isomerization are 6.7±0.6 kcal·mol −1 and 21.9±1.0 e.u., respectively.

Asymmetric synthesis of functionalized α -amino-β-hydroxy acids via chiral norephedrine-derived oxazolidines

Both anti and syn enantiomerically pure functionalized α -amino-β-hydroxy acids and derivatives were synthesized starting from norephedrine-derived oxazolidine (1). The key-steps of the synthesis were the nucleophilic epoxidation of (1) and the nucleophilic opening of epoxy acid (3) with ammonia, both reactions proved regio- and diastereospecific. High yield preparation of the target anti aldehyde (9) was accomplished using standard procedures. The complementary syn aldehyde (23) was obtained via alkaline isomerization of the cis oxazolidinone (13) to the trans one. The aldehyde function of (9) and (23) provides a useful handle for manipulation to more complex structures, allowing potential access to a range of optically pure α -amino-β-hydroxy acids. The formal total synthesis of the monocyclic β-lactam antibiotic “carumonam” was accomplished using the present methodology.

ChemInform Abstract: Alkaline Degradation of Monosaccharides. Part 5. Kinetics of the Alkaline Isomerization and Degradation of Monosaccharides

ChemInformVolume 18, Issue 25 Preparative Organic Chemistry ChemInform Abstract: Alkaline Degradation of Monosaccharides. Part 5. Kinetics of the Alkaline Isomerization and Degradation of Monosaccharides J. M. DE BRUIJN, J. M. DE BRUIJN Lab. Org. Chem., Delft Univ. Technol., 2628 BL Delft, Neth.Search for more papers by this authorKIEBOOM A. P. G. KIEBOOM A. P. G., KIEBOOM A. P. G. KIEBOOM A. P. G. Lab. Org. Chem., Delft Univ. Technol., 2628 BL Delft, Neth.Search for more papers by this authorH. VAN BEKKUM, H. VAN BEKKUM Lab. Org. Chem., Delft Univ. Technol., 2628 BL Delft, Neth.Search for more papers by this author J. M. DE BRUIJN, J. M. DE BRUIJN Lab. Org. Chem., Delft Univ. Technol., 2628 BL Delft, Neth.Search for more papers by this authorKIEBOOM A. P. G. KIEBOOM A. P. G., KIEBOOM A. P. G. KIEBOOM A. P. G. Lab. Org. Chem., Delft Univ. Technol., 2628 BL Delft, Neth.Search for more papers by this authorH. VAN BEKKUM, H. VAN BEKKUM Lab. Org. Chem., Delft Univ. Technol., 2628 BL Delft, Neth.Search for more papers by this author First published: June 23, 1987 https://doi.org/10.1002/chin.198725069Citations: 1Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume18, Issue25June 23, 1987 RelatedInformation

Alkaline degradation of monosaccharides V: Kinetics of the alkaline isomerization and degradation of monosaccharides

AbstractA new kinetic model for the alkaline isomerization and degradation of monosaccharides is presented which includes the interconversion of D‐glucose, D‐fructose, D‐mannose and D‐psicose. Computer simulations using this model fit the experimental data and allow the determination of all relevant reaction rate constants. Additionally, it has been established that, for the alkaline degradation of either D‐fructose, D‐glucose or D‐mannose, substantial amounts of acidic products, i.e. ∼65% and ∼20%, are formed via D‐fructose and D‐psicose, respectively.The influence of some reaction parameters on the pseudo‐first‐order rate constants involved in the kinetic model has been investigated. The enolization of monosaccharides appears to be the rate‐limiting step in both the isomerization and the degradation reactions. The enolization rate is markedly enhanced at higher HO− concentration or by the addition of calcium(II). Direct retro‐aldolization of D‐fructose and D‐psicose occurs at [HO−] > 10−2 M or in the presence of calcium(II), leading to an additional increase in the degradation rate towards lactic acid. The shift of the isomerization equilibrium by borate towards D‐fructose and D‐psicose is accompanied by a decrease of the degradation rate constants, due to stabilization of the monosaccharides as their borate esters.

Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c.

Titration to high pH converts yeast iso-2 cytochrome c to an inactive but more stable alkaline form lacking a 695-nm absorbance band [Osterhout, J. J., Jr., Muthukrishnan, K., & Nall, B. T. (1985) Biochemistry 24, 6680-6684]. The kinetics of absorbance-detected refolding of the alkaline form have been measured by dilution of guanidine hydrochloride in a stopped-flow instrument. Fast-folding species (tau 2) are detected, as in refolding to the native state at neutral pH. An additional kinetic phase (tau a) is observed with an amplitude opposite in sign to the fast phase. The amplitude of this phase increases and the rate increases with increasing pH. Comparison to pH-jump measurements of the fully folded protein shows that phase tau a has the same sign, rate, and pH dependence as the alkaline isomerization reaction, suggesting that this new phase involves isomerization of native or nativelike species following fast folding. Absorbance difference spectra are taken at 5-s intervals during refolding at high pH. The spectra verify that nativelike species--with a 695-nm absorbance band--are formed transiently, before conversion of the protein to the alkaline form. Refolding in the presence of ascorbate shows that the transient, nativelike species are reducible, unlike alkaline iso-2. Thus, (1) refolding to the alkaline form of iso-2 cytochrome c proceeds through transient native or nativelike species, and (2) a folding pathway leading to native or nativelike forms is maintained at high pH, where native species are no longer the thermodynamically favored product.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectrophotometric Studies on Alkaline Isomerization of Spinach Ferredoxin

The gross protein structure, the microenvironment of the iron-sulfur cluster, and the effect of neutral salts on the molecular structure of spinach ferredoxin were studied by CD and absorption spectroscopy in the alkaline pH range. In the pH range of 7-11, the existence of reversible isomerization which consisted of at least two proton dissociation processes was indicated by the statical CD and absorption spectra. The CD changes in the visible and far-UV regions were dramatic upon elevation of the pH from neutral to alkaline, indicating a significant alteration of the microenvironment of the cluster and a decrease in the ordered secondary structures. The absorption change in the visible region due to pH elevation was small but clearly observed with a high signal-to-noise ratio. The numbers of protons involved in the respective processes and the apparent pK values obtained from the pH-dependence of the CD changes were in good agreement with those obtained from the pH-dependence of the absorption changes in the visible region. In addition, the rate constants obtained from the time courses of the CD and absorption changes agreed with one another. By the addition of 1 M NaCl, the CD and absorption spectra at alkaline pH were reversed almost to those at neutral pH without significant pH change. On the other hand, above pH 11, ferredoxin was found to be irreversibly denatured. Based on analyses of the statical CD and absorption spectra and of the time courses of the CD changes, the probable mechanism of the isomerization was considered to be as follows: (Formula: see text) where H stands for a proton, N-form for native ferredoxin at neutral pH, N*-form for alkaline ferredoxin below pH 11 which still has the iron-sulfur cluster but with disordered secondary structures of the polypeptide chain, and D-form for completely denatured ferredoxin above pH 11. These results lead to the conclusions that (1) the interaction between the protein moiety and the iron-sulfur cluster is essential for maintaining the native ferredoxin structure, and (2) neutral salts protect the polypeptide chain from unfolding through electrostatic interaction with the ionized side chains, resulting in the stabilization of ferredoxin.

Time-resolved resonance Raman study of alkaline isomerization of ferricytochrome c

Time-resolved resonance Raman study of alkaline isomerization of ferricytochrome c

Metabolism of arachidonate in rat testis: characterization of 26-30 carbon polyenoic acids.

Fatty acid methyl esters of long-chain polyenoic fatty acids (LCPA) from rat testis injected with [1-14C] arachidonate were analyzed and separated by reversed-phase high performance liquid chromatography (RP-HPLC). Earlier, all previously identified LCPA were prepared in high purity along with 4 previously unidentified fatty acids, which were further characterized by capillary gas chromatography (GC), catalytic hydrogenation and alkaline isomerization. Unidentified fatty acids proved to be 26:4, 26:5, 28:5 and 30:5. All of these LCPA incorporated 14C from arachidonate (20:4) to specific activities that were comparable to that of 20:4 and previously identified metabolites of 20:4 and much greater than specific activities of 18:1n-9 or 22:6n-3. LCPA were analyzed on a capillary GC system capable of resolving known cis-trans and positional isomers of the n-3, n-6, n-7 and n-9 families of unsaturated fatty acids. Log plots of isothermal retention times and normal plots of temperature programmed retention times were linear (r = 0.999) in carbon number when values for known and previously unidentified LCPA of 4 or 5 double bonds, respectively, were coplotted. Thus, the newly identified fatty acids belong to the n-6 family of fatty acids synthesized from arachidonic acid.

Evidence for sulphur ligation in ferricytochrome c at alkaline pH

The inactivation of ferricytochrome c at alkaline pH proceeds with a pK of 9.3 and it has been shown that the process involves a slow conformational change coupled to a real ionization with pK of 11 [1]. Important indicators of the alkaline isomerization are the disappearance of the ironsulfur charge transfer band at 695 nm [2], and the disappearance and formation of signals monitored by NMR [3] and EPR [4]. The prevalent view has been that Met-80, the sixth ligand, is replaced by another strong field ligand [3, 4]. However, the results described below strongly suggest that Met-80 remains ligated to the haem iron. The 1H NMR signals (270 MHz) arising from the haem methyl groups of the first alkaline form [3] are found to be composed of overlapping resonances from two slowly exchanging forms of ferricytochrome c. Either form can be made dominant over the other by raising the temperature to 330 K or by addition of 0.3 M perchlorate at pH 10. In the upfield region of the spectrum two very broad resonances between −8 and 10 ppm are assigned to the Met-80 methyl group in its two environments. Modification of Met-80 by carboxymethylation [3, 4] or methylation perturbs the equilibrium constant for the conformation change involved in the alkaline isomerization so that it can be observed in the neutral pH range. In the latter case just one ‘alkaline’ form is observed by NMR and the resonances assigned to Met-80 now show up with twice the intensity for the corresponding peaks in the unmodified protein. Also in the carboxymethylated form of ferricytochrome c the Met-80 methyl resonances are clearly visible in the same ppm range. The NMR results were correlated with the signals appearing at alkaline pH in EPR spectra (9.2 GHz and 17 K). The two forms observed by NMR thus correspond to the g z = 3.39 and g z = 3.57 signals. An additional EPR species with g z = 3.26 was correlated with the form of ferricytochrome c where the conformation change, but not the deprotonation of the group with pK = 11, has occurred. This form of cytochrome c can also be observed at high temperatures [5] or high urea concentrations [6] at neutral pH. As expected both carboxymethylation and methylation of Met-80 yielded the g z = 3.26 species as the major form at neutral pH. The occurrence of two alkaline forms, as described above, is most likely due to a slow exchange between the two chiral binding modes of Met-80 to the haem iron. This is also strongly supported by the modification experiments on Met-80. The anomalously broadened resonances stemming from the Met-80 methyl group (the width at half height is approximately 350 Hz) are probably due to the slow flipping rate of the nearby Tyr-67. Such an exchange can lead to appreciable broadening since the scalar relaxation of the Met-80 methyl resonance, due to the haem iron, will be modulated by the exchange rate [7]. Considering the positive charge on the sulphur atom of Met-80 in the modified proteins, it is likely that repulsion between the sulphur and the iron will lead to an increased ironsulphur bond length. This would rapidly explain the disappearance of the 695 nm band, the decreased hyperfine shifts in NMR as well as the EPR signals appearing at alkaline pH.

Kinetics of alkali-catalyzed isomerization of D-glucose and D-fructose in ethanol-water solutions

The kinetics of the alkaline isomerization of D-glucose and D-fructose in 0.1 M ethanol-water solutions of sodium hydroxide were studied. To calculate the hydroxylion-independent rate-constants, the ionization equilibria of the respective sugar were determined separately by potentiometric titration in the same solvent system at 21°. The sugars ( D-glucose and D-fructose) in the reaction mixture were analyzed by g.l.c. as the trimethylsilylated oximes. By the micro-scale technique developed, reliable data could be obtained for reactions shorter than 10 seconds. The isomerization of D-glucose to D-fructose, and vice versa, accelerated markedly as a function of the alcohol concentration, and in 70% ethanol, their rates were 2.4 and 1.7 times the corresponding values in water solution. The activation energies of these isomerization reactions were found to be 122 kJ.mol −1 ( D-glucose) and 112 kJ.mol −1 ( D-fructose), and they were independent of the ethanol concentration.

Alkaline isomerization of ferricytochrome c: Lysine is not replacing methionine at the sixth co-ordination site of the haem iron

Oxidized cytochrome c is known to undergo a restricted conformational refolding of its haem area at around pH 9. Methionine 80, the sixth ligand of the ferric haem iron in the biologically active neutral conformational state, is replaced by a new strong-field ligand in the biologically inactive alkaline state of the molecule. It had been proposed that a lysine residue, possibly lysine 79. is the new haem ligand. We have tested this proposition by a more direct approach than hitherto employed, namely by measuring the relative chemical reactivity of lysines in the oxidized eytochrome c and in fragment 66–80 cut out of the native molecule. The relative rates of acetylation of lysine 79, measured between pH 7 and pH 11, are virtually identical in the intact molecule and in the haem-free fragment 66–80. Similarly, the rates are also the same for the amidination reaction with isethionylacetimidate. When the relative rates of acetylation and amidination of lysines 72 + 73 were compared there was again no significant difference between the intact molecule and fragment 66–80. These results contradict the involvement of any of the three lysines in the alkaline isomerization, as a haem-bound ϵ-amino group would be much less reactive than its freely accessible counterpart in fragment 66–80. To corroborate the above finding, the p K value and absolute rate constant of acetylation of lysine 79 were determined and compared with the respective values for lysines 39 and 60. The latter two residues are on the side opposite to the haem pocket and hence unable to bind to the haem iron. The three p K values and rate constants k obey the Brønsted relationship: log κ = α + βp K with β = 0.48, a value characteristic of the acetylation of freely accessible primary amino groups. Taken together, these results oppose an ϵ-amino: haem iron co-ordination in the alkaline state of oxidized eytochrome c.

Kinetic and equilibrium studies of alkaline isomerization of vertebrate cytochromes c

Equilibria and kinetics of alkaline isomerization of seven ferricytochromes c from vertebrates were studied by pH-titration and pH-jump methods in the pH region of 7–12. In the equilibrium behavior, no significant difference was detected among the cytochromes c, whereas marked differences in the kinetic behavior were observed. According to the kinetic behavior of the isomerization, the cytochromes c examined fall into three classes: Group I (horse, sheep, dog and pigeon cytochromes c), Group II (tuna and bonito cytochromes c) and Group III (rhesus monkey cytochrome c). The kinetic results are interpreted in terms of the sequential scheme: ▪ where the neutral and alkaline forms are the species stable at neutral and alkaline pH, respectively, and the transient form is a kinetic intermediate. From comparison of the primary sequences of the seven cytochromes c and the classification of these cytochromes c, it is concluded that the amino acid substitution Phe/Tyr at the 46-th position has a major influence on the kinetic behavior. In Group II and III cytochromes c, the ionization of Tyr-46 is suggested to bring about loosening of the heme crevice and thus facilitate the ligand replacement involved in the isomerization.

A Transient Spin-State Change during Alkaline Isomerization of Ferricytochrome c

Kinetic difference spectra during the alkaline isomerization of ferricytochrome c were obtained by the pH-jump method in the range of 540 to 655 nm. The spectrum of the transient intermediate, which appears during the course of the isomerization, was reproduced from the spectra. The intermediate showed an intense absorption band at 600 nm, indicating that it is a high spin or mixed spin species. This is in contrast to the stable neutral and alkaline forms which are low spin species. The transient spin-state change during the isomerization was also observed upon rapid oxidation of ferrocytochrome c at alkaline pH.

Electron transfer reactions of cytochrome f from Brassica komatsuna with hexacyanoferrate.

The electron transfer reactions of membrane-bound monomeric cytochrome f from Brassica komatosuna (Brassica rapa L. var. perviridis Bailey) with hexacyanoferrate (II)-(III) have been studied as a function of pH, ionic strength and temperature. The second-order rate constant for the oxidation of cytochrome f by Fe(CN)6(3-) at pH 7.0, mu 0.1 M, and 20 degrees C is 1.7 X 10(5) M-1 X S-1, which is similar to the value of oligomeric cytochrome f from parsley. The activation parameters obtained were delta H not equal to = -0.87 kcal/mol and delta S not equal to - -38 cal/mol x deg. Respective rat constant and activation parameters obtained for the reduction of cytochrome f by Fe(CN)6(4-) were k = 1.7 X 10(4) M-1 x S-1, delta H not equal to = +6.7 kcal/mol, and delta S not equal to -16 cal/mol x deg. Both the rate constants for the oxidation and the reduction of cytochrome f markedly decreased with increasing ionic strength. The results indicate that the oxidation and the reduction take place at a positively charged site on the cytochrome f surface, and electrostatic interactions are important for these reactions. The participation of protons and specific amino acid residues in electron transfer reactions of cytochrome f is implied from the pH results. Alkaline isomerization of ferricytochrome f was not observed. The midpoint potential of cytochrome f has a constant value of 360 mV between pH 5.0-8.9, and decreases by about 55 mV per pH unit above 8.9. The results are compared with the data for horse heart cytochrome c and Euglena gracilis cytochrome c-552. These data are discussed in relation to the theories of electrostatic corrected outer-sphere electron transfer of Marcus and multiphonon nonadiabatic electron tunneling of Jortner and Hopfield.

Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8.

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.