Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pKa values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pKa value and the corresponding experimental pKa value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.     

Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR

The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand tau nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine phi nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine tau NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the omega loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absorption and Raman spectroscopy study of cyt c-thiol complexes in acidic solutions.

Absorption UV-VIS and pre-resonance Raman spectra of acidic cyt c solutions with a series of thiols added (thiophenol, n-propanethiol, isopropanethiol, L-cysteine, dithiothreitol, 2-mercaptoethanol, N-acetyl-L-cysteine, p-acetamidothiophenol, 2-mercaptoethanamine, thioglycolic acid and mercaptopropionic acid), are presented. Interactions of cyt c molecule with the thiols were studied with the aim to identify binding of the thiols with the cyt c heme as its iron axial ligands. Absorption and Raman spectra showed some correlation between maxima of 700 nm region absorption band (typical for Fe-S axial bond in cyt c heme) and also wave numbers of spin state marker and axial ligand sensitive Raman bands on one, and pKa constant values of appropriate thiols on the other hand. These results imply thiol replacement of Met-80 from axial bond with heme iron and suggest that the force of Fe-L-cysteine axial bond is very close to the native axial bond (Fe-Met) for cyt c in neutral solution.     

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.     

Sequential conformation change and activation of chicken liver dihydrofolate reductase in low concentration of guanidine hydrochloride

The conformation changes of dihydrofolate reductase (DHFR) from chicken liver in guanidine hy-drochloride were monitored by protein intrinsic fluorescence, hydrophobic fluorescence probe TNS and limited proteol-ysis by proteinase K. The kinetics of the enzyme denaturation were also studied and compared with its activity changes. It was indicated by the enhanced fluorescence of 2-p-toluidinylnaphthalene (TNS) that a subtle conforma-tional change of the enzyme in dilute GuHCl parallels GuHCl-induced activation. At GuHCl concentration higher than 0.75 mol/L, the conformational change can be detected by increased susceptibility of the enzyme to proteinase K, but no significant gross conformational change of the enzyme molecule is observed by intrinsic fluorescence up to a GuHCl concentration of 1.2 mol/L. The results suggest that the denaturation of DHFR by GuHCl does not follow strictly the two-state model. The enzyme seems to open up sequentially with increasing concentrations of denaturants, mainly at th     

Alkaline isomerization of horse and yeast cytochromes C. Spectrophotometric and circular dichroism studies.

Spectrophotometric studies of the alkaline isomerization of horse heart and yeast cytochrome c show that the haemoproteins from Saccharomyces cerevisiae differ significantly from the mammalian cytochrome c. Apparent pKa values of 8.41, 8.40 and 8.73 for isol-1-(the methylated and unmethylated forms) and iso-2-cytochrome c respectively, from baker's yeast were determined and compared with the value of 9.40 found for horse heart cytochrome c. The transitions, measured by observing the decrease of the absorbance at 695 nm as the pH increases, have been found to strictly parallel the decrease in amplitude of the negative circular dichroism band centered at 417 nm. This observation gives additional evidence that this negative band is closely related to the ligation of the heme iron by the sulfur atom of methionine 8u for each of the four haemoproteins examined.     

Heme coordination states of unfolded ferrous cytochrome C

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.     

NMR studies of the conformations of leghemoglobins from soybean and lupin

Phase-sensitive two-dimensional NMR methods have been used to obtain extensive proton resonance assignments for the carbon monoxide complexes of lupin leghemoglobins I and II and soybean leghemoglobin a. The assigned resonances provide information on the solution conformations of the proteins, particularly in the vicinity of the heme. The structure of the CO complex of lupin leghemoglobin II in solution is compared with the X-ray crystal structure of the cyanide complex by comparison of observed and calculated ring current shifts. The structures are generally very similar but significant differences are observed for the ligand contact residues, Phe30, His63 and Val67, and for the proximal His97 ligand. Certain residues are disordered and adopt two interconverting conformations in lupin leghemoglobin II in solution. The proximal heme pocket structure is closely conserved in the lupin leghemoglobins I and II but small differences in conformation in the distal heme pocket are apparent. Larger conformational differences are observed when comparisons are made with the CO complex of soybean leghemoglobin. Altered protein-heme packing is indicated on the proximal side of the heme and some conformational differences are evident in the distal heme pocket. The small conformational differences between the three leghemoglobins probably contribute to the known differences in their O2 and CO association and dissociation kinetics. The heme pocket conformations of the three leghemoglobins are more closely related to each other than to sperm whale myoglobin. The most notable differences between the leghemoglobins and myoglobin are: (a) reduced steric crowding of the ligand binding site in the leghemoglobins, (b) different orientations of the distal histidine, and (c) small but significant differences in proximal histidine coordination geometry. These changes probably contribute to the large differences in ligand binding kinetics between the leghemoglobins and myoglobin.     

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43.     

Ligand and halide binding properties of chloroperoxidase: peroxidase-type active site heme environment with cytochrome P-450 type endogenous axial ligand and spectroscopic properties

Equilibrium binding studies of exogenous ligands and halides to the active site heme iron of chloroperoxidase have been carried out from pH 2 to 7. Over twenty ligands have been studied including C, N, O, P, and S donors and the four halides. As judged from changes in the optical absorption spectra, direct binding of the ligands to the heme iron of ferric or ferrous chloroperoxidase occurs in all cases; this has been ascertained for the ferric enzyme in several cases through competition experiments with cyanide. All of the ligands except for the halides, nitrate, and acetate form exclusively low-spin complexes in analogy to results obtained with the spectroscopically related protein, cytochrome P-450-CAM [Sono, M., & Dawson, J.H. (1982) J. Biol. Chem. 257, 5496-5502]. The titration results show that, for the ferric enzyme, (i) weakly acidic ligands (pKa greater than 3) bind to the enzyme in their neutral (protonated) form, followed by deprotonation upon ligation to the heme iron. In contrast, (ii) strongly acidic ligands (pKa less than 0) including SCN-, NO3-, and the halides except for F- likely bind in their anionic (deprotonated) form to the acid form of the enzyme: a single ionizable group on the protein with a pKa less than 2 is involved in this binding. For the ferrous enzyme, (iii) a single ionizable group with the pKa value of 5.5 affects ligand binding. These results reveal that chloroperoxidase, in spite of the previously established close spectroscopic and heme iron coordination structure similarities to the P-450 enzymes, clearly belongs to the hydroperoxidases in terms of its ligand binding properties and active site heme environment. Magnetic circular dichroism studies indicate that the alkaline form (pH 9.5) of ferric chloroperoxidase has an RS-ferric heme-N donor ligand coordination structure with the N donor likely derived from histidine imidazole.     

Cytochrome c at charged interfaces. 1. Conformational and redox equilibria at the electrode/electrolyte interface probed by surface-enhanced resonance Raman spectroscopy

The structure and the electron-transfer properties of cytochrome c (cyt c) absorbed on a silver electrode were analyzed by surface-enhanced resonance Raman spectroscopy. It was found that the absorbed cyt c exists in various conformational states depending on the electrode potential. In state I the native structure of the heme protein is fully preserved and the redox potential (+0.02 V vs saturated calomel electrode) is close to the value for cyt c in solution. In state II the heme iron exists in a mixture of five-coordinated high-spin and six-coordinated low-spin configurations. It had been shown that these configurations form a thermal equilibrium [Hildebrandt, P., & Stockburger, M. (1986) J. Phys. Chem. 90,6017]. It is demonstrated that these equilibria strongly depend on the charge distribution within the electrical double layer of the silver electrode/electrolyte interface, indicating that the changes in the coordination shell are induced by electrostatic interactions. The structural alterations in state II are apparently restricted to the heme crevice, which assumes an open conformation compared to the close structure in state I. This leads to a strong decrease of the redox potentials, which were determined to be -0.31 and -0.41 V for the five-coordinated high-spin and six-coordinated low-spin configurations, respectively. On the other hand, gross distortions of the protein structure can be excluded since the reversible proton-induced conformational change of cyt c as found in solution at low pH also takes place in state II of the absorbed cyt c. The linkage of cyt c molecules to the surface is mediated by charged amino acid groups, and it depends on the potential which groups are thermodynamically favored to form such a molecular binding site. The conformational states I and II, which are in potential-dependent equilibrium, therefore refer to two different molecular binding sites. At potentials below zero charge (less than approximately -0.6 V) a rapid denaturation of the absorbed cyt c is noted, which is reflected by drastic and irreversible changes in the surface-enhanced resonance Raman spectrum. Our results are discussed on the background of previous electrochemical studies of cyt c at electrodes.     

Unfolding and flexibility in hemoproteins shown in the case of carboxymethylated cytochrome c

A circular dichroism study of carboxymethylated cytochrome c has been performed to obtain further information on the structural basis responsible for the observed changes in ligand binding and redox properties of the modified cytochrome c. The results give additional evidence of local structural changes occurring in the heme environment upon rupture of the (Met-80)-heme iron bond in the modified protein. This produces no alterations of the overall molecular conformation, but results in drastic changes in redox potential. In addition, analysis of the reversible conformational transitions induced by urea in the native and the modified proteins supports the idea that the modified derivative can be considered as an 'intermediate state' between the native and the fully unfolded protein.     

Contribution of protein conformation to stereochemistry and reactivity of the active center of heme proteins and enzymes. The existence of horseradish peroxidase conformations and their possible role in the catalysis mechanism

In the spectral region 350-800 nm at 4.2 K we measured magnetic circular dichroism (MCD) spectra of the pentacoordinated complex of protcheme with 2-methylimidazole, deoxyleghemoglobin, neutral and alkaline forms of reduced horseradish peroxidase in the equilibrium states, as well as in non-equilibrium states produced by low-temperature photolysis of their carbon monoxide derivatives. Earlier the corresponding results have been obtained for myoglobin, hemoglobin and cytochromes P-450 and P-420. The energies of Fe-N (proximal His) and Fe-N(pyrroles) bonds and their changes upon ligand binding in heme proteins and enzymes were compared with those in the model heme complex thus providing conformational contribution into stereochemistry of the active site. The examples of weak and strong conformational "pressure" on stereochemistry were analysed and observed. If conformational energy contribution into stereochemistry prevails the electronic one the heme stereochemistry remains unchanged on ligand binding as it was observed for leghemoglobin and alkaline horseradish peroxidase. The change of bond energies in myoglobin and hemoglobin on ligand binding are comparable with those in protein free pentacoordinated protoheme, giving an example of weak conformational contribution to heme stereochemistry. The role of protein conformation energy in the modulation of ligand binding properties of heme in leghemoglobin relative to those in myoglobins is discussed. The most striking result were obtained in the study of reduced horseradish peroxidase in the pH region of 6.0-10.2. It was found that such different perturbations as ligand binding and heme-linked ionization of the distal amino acid residue induce identical changes in heme stereochemistry. Neither heme-linked ionization in the carbon monoxide complex nor the geometry of Fe-Co bond affect the heme local structure of photoproducts. These and other findings suggest a very low conformation mobility of horseradish peroxidase whose protein constraints appear to allow only two preferable geometries of specific amino acid residues that form the heme pocket. The role of the two tertiary structure constraints on the heme in the mechanism of horseradish peroxidase function is discussed. It is supposed that one conformation produces a heme environment suitable for two-electron oxidation of the native enzyme to compound I by hydrogen peroxide while another conformation changes the heme stereochemistry in the direction favourable for back reduction of compound I by the substrate to the resting enzyme through two one-electron steps. The switch from one tertiary structure to another is expected to be induced by substrate bind     

Optical spectroscopic differentiation of various equilibrium denatured states of horse cytochrome c

Thermal unfolding of cytochrome c (cyt c) from several states has been studied using equilibrium spectroscopic techniques. CD in the uv, vibrational circular dichroism, infrared, and uv-vis absorption spectra measured at various temperatures, pHs, salt concentrations, and GuHCl concentrations are used to show the conformational as well as heme structural differences between native and various denatured states. The difference in thermal denaturation behaviors of cyt c starting from acid denatured, molten globule (MG), and the A and native states are explored. Different final high temperature states were observed for cytochrome c unfolding from four different initial states (native, MG, A, and acid denatured state) by electronic CD, Fourier transform infrared (FTIR), and vibrational CD (VCD). Consistent with this, different thermal unfolding pathways for the MG and A states are suggested by the FTIR and VCD data for this process.     

The guanidine hydrochloride-induced denaturation of CP43 and CP47 studied by terahertz time-domain spectroscopy

Terahertz time-domain spectroscopy (THz-TDS) is a new technique in studying the conformational state of a molecule in recent years. In this work, we reported the first use of THz-TDS to examine the denaturation of two photosynthesis membrane proteins: CP43 and CP47. THz-TDS was proven to be useful in discriminating the different conformational states of given proteins with similar structure and in monitoring the denaturation process of proteins. Upon treatment with guanidine hydrochloride (GuHCl), a 1.8 THz peak appeared for CP47 and free chlorophyll a (Chl a). This peak was deemed to originate from the interaction between Chl a and GuHCl molecules. The Chl a molecules in CP47 interacted with GuHCl more easily than those in CP43. Supported by the National Natural Science Foundation of China (Grant No. 39890390)     

pH-induced conformation changes and equilibrium unfolding in yeast iso-2 cytochrome c

The relationship between pH-induced conformational changes in iso-2 cytochrome c from Saccharomyces cerevisiae and the guanidine hydrochloride induced unfolding transition has been investigated. Comparison of equilibrium unfolding transitions at acid, neutral, and alkaline pH shows that stability toward guanidine hydrochloride denaturation is decreased at low pH but increased at high pH. In the acid range the decrease in stability of the folded protein is correlated with changes in the visible spectrum, which indicate conversion to a high-spin heme state--probably involving the loss of heme ligands. The increase in stability at high pH is correlated with a pH-induced conformational change with an apparent pK near 8. As in the case of homologous cytochromes c, this transition involves the loss of the 695-nm absorbance band with only minor changes in other optical parameters. For the unfolded protein, optical spectroscopy and 1H NMR spectroscopy are consistent with a random coil unfolded state in which amino acid side chains serve as (low-spin) heme ligands at both neutral and alkaline pH. However, the paramagnetic region of the proton NMR spectrum of unfolded iso-2 cytochrome c indicates a change in the (low-spin) heme-ligand complex at high pH. Apparently, the folded and unfolded states of the (inactive) alkaline form differ from the corresponding states of the less stable native protein.     

Ligand binding-promoted conformational changes in yeast ornithine transcarbamoylase

It has been proposed that regulatory multienzyme complex formation between yeast ornithine transcarbamoylase (OTCase) and arginase is triggered by a conformational change promoted by the binding of ornithine to a regulatory site in OTCase (Wiame, J.-M. (1971) Curr. Top. Cell. Regul. 4, 1-38). To isolate the binding of ornithine to the proposed regulatory site, the active site was blocked with the high affinity (Ki = 13 +/- 1.4 nM) bisubstrate analogue, delta-N-phosphonacetyl-L-ornithine (PALO). The binding of PALO to the active site produces large changes in the absorption (delta A290-296 = 0.010/mg of enzyme) and in the fluorescence (25% quenching) of the protein. These changes both saturate at one PALO/polypeptide chain. The binding of PALO also changes the rate constant for diffusional acrylamide quenching by 43% and increases the midpoint for the thermal denaturation of the enzyme by 13 degrees C. Finally, PALO binding results in a +2.8% change in the sedimentation coefficient demonstrating that these spectral and energetic changes are associated with a gross structural change in the enzyme. In an effort to detect ligand binding to the proposed effector site on OTCase, ornithine was added to the enzyme saturated with PALO, and consequent conformational changes were tested for using methodologies identical to those which demonstrated active site ligand binding-promoted conformational changes. In no instance were any additional differences observed. Hence, strong support for isosteric effector binding-promoted conformational changes cannot be presented. We conclude that active site ligand binding events themselves are responsible for conformational changes which promote enzyme-enzyme association of OTCase with arginase.     

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.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.