NMR OF HEME CARBONYLS
Carbon- 13 Nuclear Magnetic Resonance of Heme Carbonyls. Cytochrome c and Carboxymethyl Derivatives of Cytochrome c t L. 0. Morgan,*.i R. T. Eakin, P. J. Vergamini, and N. A. Matwiyoff
ABSTRACT: Carbonyl complexes of horse cytochrome c and various carboxymethylated derivatives have been examined using I3C N M R (carbon- 13 nuclear magnetic resonance) spectroscopy. The multiplicity and chemical shift of the I3CO resonance were found to be functions of pH and the extent of alkylation. Correlations have been made among prominent
c
ytochrome c is a low molecular weight (1 2 400 daltons) heme protein whose tertiary structure has been highly conserved throughout evolution. The structural similarity of the cytochromes c among a wide variety of plant and animal species is indicative of the critical importance of the electronic environment of the heme group in the functional role of this protein as a biological electron carrier. Thus, the determination of the electronic environment and protein configuration in the vicinity of the heme of cytochrome c is of particular importance in understanding the mechanism by which this protein transfers electrons in biological oxidation processes. One of the classical methods of probing the electronic environment of heme in heme proteins is through the formation of complexes with small ligands such as carbon monoxide, molecular oxygen, or nitrous oxide. Visible and infrared spectroscopy of such complexes have given some indication of the structural and electronic nature of the heme environment, and recently formation of carbonyl complexes with 13C0has provided a more sensitive probe via 13C N M R (carbon-13 nuclear magnetic resonance) spectroscopy. This method has shown differences, for example, among the heme environments of several heme protein-CO complexes, including sperm whale myoglobin (Moon and Richards, 1972a) and the CY and 0 chains of various species of hemoglobins (Matwiyoff et al., 1973; Vergamini et al., 1973; Moon and Richards, 1972a,b). In the native state of cytochrome c, the histidyl-18 and the methionyl-80 residues of the protein serve as axial ligands for the heme group and prevent formation of carbonyl complexes. However, procedures which remove one or both of these axial ligands, such as carboxymethylation in the presence of cyanide (Schejter and Aviram, 1970), lowering the pH (Cohen et al., 1974), or denaturing in urea or guanidine hydrochloride (Knapp and Pace, 1974), allow the formation of carbonyl derivatives. We have investigated the carbonyl complexes of some of these derivatives using 13CN M R spectroscopy and, in the From the Department of Chemistry, The University of Texas at Austin (L.O.M.and R.T.E.),Austin, Texas 78712, and The Los Alamos Scientific Laboratory (P.J.V. and N.A.M.), University of California, Los Alamos, New Mexico 87545. Received November 18. 1975. This work was performed under the auspices of the United States Energy Research and Development Administration and was supported in part by the Robert A. Welch Foundation, Houston, Texas. University of Texas at Austin, University Research Institute grantin-aid.
*
features of the chemical shift titration curves and changes in the environment of the heme. A simple model compatible with the bulk of previous observationshas been suggested to account for the several carbonyl resonance peaks and the complex behavior of the chemical shift with changes in pH.
results presented here, describe pH-dependent conformational transitions and heme environment heterogeneity. Materials and Methods Sources of Materials. All alkylated derivatives of cytochrome c were prepared from horse heart cytochrome c (Sigma Chemical Company, type VI). Natural abundance carbon (1.1 atoms %) bromoacetic acid was obtained from Eastman Kodak. [2-13C]Br~m~acetic acid used in some of the carboxymethylation reactions and [ 12C]urea used for denaturing cytochrome c were a gift from Donald Ott and Vernon Kerr of the Los Alamos Scientific Laboratory. Enriched I3CO (90+ atoms %) was a gift from B. B. McInteer of the Los Alamos Scientific Laboratory. Preparation of Carboxymethylcytochrome c. Carboxymethyl derivatives of cytochrome c were prepared in several different ways, as previously reported (Eakin et al., 1975). In that work, estimates of carboxymethyl product formation in the various types of amino acid residues from alkylation with [2-13C]bromoacetate were obtained from 13C N M R spectra of the modified proteins. Major features of the alkylation processes were found to conform with those obtained in other laboratories, although evidence for glycolate ester formation and more extensive lysyl modification appeared in the 13C NMR spectroscopic analyses. Three classes of carboxymethyl derivatives used in this work may be described as follows: (1) Dicarboxymethylmethionylcytochromec [(CmMet)2c;' Ando et al., 1965; Tsai and Williams, 1965; Babul and Stellwagen, 1972)] was formed from acid denatured cytochrome c at pH 3. Both methionyl residues were alkylated and essentially no other residues were affected. (2) Carboxymethylcytochrome c from a 6-day alkylation reaction time [(6dCm)c; Harbury et al., 1965; Stellwagen, 1968; Schejter apd Aviram, 1970)] was formed at pH 7 from cyanoferricytochrome c in 0.1 M CN-. This preparation contained 2 alkylated methionyl residues, 1 alkylated histidyl residue, 2 alkylated lysyl residues, and 2.6 glycolate ester products. (3) Carboxymethylcytochrome c from a 1-day alkylation reaction time [(ldCm)c] was formed at pH 7 from cyanoferricytochrome c in 0.1 M CN-. I Abbreviations used: (CmMet)Zc, dicarboxymethylmethionylcytochrome c; (6dCm)c, carboxymethylcytochromec from a 6-day alkylation reaction time; (ldCm)c, carboxymethylcytochrome c from a I-day alkylation reaction time.
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f
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1
I
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1
1
I
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4
5
6
7
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FIGURE 1: Chemical shift titration curves for I3COcomplexes of cytochrome c derivatives; parts per million downfield from Me&i as a function of solution pH. (a) Unmodified cytochrome c; (b) (1dCm)c; (c) (6dCm)c; (d) (CmMet)zc.
The alkylation was less extensive than in the analogous 6-day reaction time preparation, amounting to the formation of 1.3 carboxymethylmethionyl residues, ca. 0.6 carboxymethylhistidyl residues, ca. 0.8 carboxymethyllysyl residues, and 0.5 glycolate ester products. On the basis of previous kinetic results (Stellwagen, 1966, 1968), we estimate that 0.9 methionyl-80 and 0.4 methionyl-65 residues were carboxymethylated, to give the observed total of 1.3. In both (1dCm)cl and (6dCm)c, dicarboxymethylation of histidyl and lysyl residues was observed and the estimated amounts are included in the specified totals. The I3C-labeled methylene group magnetic resonance peaks for monocarboxymethyllysyl and monocarboxymethylhistidyl residues could not be resolved so that the relative abundances of those modified residues listed above represent an estimate based on previous observations (Harbury, 1966; Stellwagen, 1966, 1968). Preparation of Carbonyl Derivatives of Carboxymethylcytochrome c. Carbonyl derivatives of the various preparations of carboxymethylcytochrome c were obtained by reduction of aqueous solutions with sodium dithionite. Without removing the dithionite, the solutions were saturated with I3CO from slow bubbling at atmospheric pressure for at least 10 min. After obtaining I3CO spectra on some solutions, a chemical shift titration was performed by sequential adjustment of pH with phosphoric acid, hydrochloric acid, or concentrated potassium hydroxide solution, treatment with dithionite, and resaturation with 13C0. N M R Spectroscopy. Fourier transform 3 C 0 NMR spectra were obtained at 20 OC as described previously (Eakin et al., 1975) over a 200-Hz region using 1024 data points in the time domain, with a total sampling time of 2.56 s. Although T I values for I3CO bound to iron(I1) in hemoglobins are 1.1- 1.2 s, corresponding values of T2 are very short and observed lines are not appreciably saturated. Chemical shift values were calculated in parts per million downfield relative to neat tet-
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FIGURE 2: Carbonyl resonance spectra of I3CO complexes of cytochrome 5; (b) (ldCm)c, pH 7; (c) (6dCm)c. pH
c derivatives. (a) (ldCm)c, pH
I.
ramethylsilane ( M e S i ) measured under similar conditions. Spectra were analyzed by least-squares line-shape analysis of the full transformed data set, utilizing a Fortran-based computer program which resolves complex line shapes into Lorentzian components. Chemical shifts of the peak centers and relative peak areas, along with their standard deviations, are calculated from the resolved curves as parts of the program. Results Chemical shifts of carbonyl resonances were measured over the pH range 2-9 for Fe(II)-I3CO complexes of unmodified cytochrome c (Figure la), (1dCm)c (Figure lb), (6dCm)c (Figure IC), and (CmMet)2c (Figure Id). Reduction of unmodified cytochrome c in aqueous solution with sodium dithionite, followed by saturation of the solution with 13C0did not produce a detectable carbonyl complex above pH 5. At pH 4.73 a weak resonance peak appeared at 205.40 ppm. Progressive lowering of the pH resulted in the chemical shift titration curve shown in Figure la, with peak intensity reaching a maximum in the pH range 3-4. No significant differences were observed between shifts observed in samples adjusted with phosphoric acid and with hydrochloric acid. Similar treatment of the three different carboxymethylcytochrome preparations yielded complexes exhibiting carbonyl resonances over the entire experimental range, pH 2-9. Representative spectra are shown in Figure 2. In [13CO]-(ldCm)c two prominent components appeared (Figure 2b), with peak positions indicated as separate branches of the titration curve (Figure lb) above pH 5. At lower pH values a single resonance peak was observed (Figure 2a) for which the spectral position and pH dependence were essentially the same as those found for [13CO]-(6dCm)c (Figure IC) and [13CO]-(CmMet)2c (Figure Id). A single resonance was observed at 205.55 ppm when the pH was increased to 11 .O. Subsequent adjustment of the solution pH to 7.1 caused a reappearance of the two-peak system. The carbonyl complex of (6dCm)c produced a spectrum comprising a single principal peak, although a small
N M R OF HEME CARBONYLS
shoulder, 0.8 ppm upfield from the peak maximum, was discernible (Figure 2c) at all except the lowest pH values, where it could easily have been masked by line broadening, or lost in the noise. In general, the behavior of the shoulder followed that of the main peak and maintained a constant relative amplitude over the pH range, where it was observable. For intermediate alkylation periods, with other variables held constant, the derivatized cytochromes gave carbonyl complexes displaying two-component spectra in the carbonyl region. Chemical shifts of the two components were the same as those in (1dCm)c and relative intensities varied in a linear fashion between the 1- and 6-day extremes. At reaction times of 1, 2.5, and 6 days, the fractional intensities of the upfield peak were 0.35, 0.55, and 1.0, respectively. In the first case, the relative intensity of the upfield peak was observed to increase to ca. 0.5 upon decrease of pH to 5.3. On the basis of total peak integrals and observed signalto-noise ratios obtained under comparable spectrometric conditions, it appeared that for the carboxymethyl derivatives, C O coordination to the heme group was optimal in the pH range 5-7. Because of variations in both protein concentration and ionic strength in the usual titration procedure, such a comparison is qualitative, at best. An attempt was made to improve the accuracy as follows. A sample of (1dCm)c was desalted on a Sephadex G-25 column, freeze-dried, and dissolved at 100 mg/ml concentration in four potassium phosphate solutions of differing pH, but constant phosphate concentration (1 .OO M). After dithionite reduction and saturation with I3CO gas, final pH values were 4.47,5.00,5.47, and 8.30. With constant spectrometer settings and with the same numbers of rf pulses, total integrated intensities were found to be 0.64,0.9 1, 1.OO, and 0.78, respectively, normalized to the peak intensity of the pH 5.47 solution. Discussion The experimental results clearly establish that the chemical shift of the I3CO resonance in carbonyl complexes of carboxymethylcytochrome c is sensitive to both pH and extent of protein alkylation. Although the titration curves of Figure 1 appear complex, the data can easily be correlated with reasonable changes in the electronic environment at the heme binding site due to variation in pH or alkylation of amino acid side chains at critical positions. At extremes of pH the protein chain is likely to be unfolded to an extent that the hydrophobic cleft is replaced by a more hydrophilic environment dominated by solvent water. In such cases the extent of alkylation of the protein would not be expected to have a significant effect on the chemical shift position of the heme bound carbonyl resonance, although it might affect details of the protein conformation. I n intermediate pH regions where the tertiary protein structure is still in its compact globular state, carbonyl complexes will be observed only when the methionyl-80 residue has been displaced from its axial coordination position, as by carboxymethylation. The modification of other proximal amino acid side chains could be expected to influence the position of the carbonyl resonance in this pH range where the protein chain can contribute substantially to the electronic environment of the heme group. Two such additional modifications which can be anticipated from previous observations by Stellwagen (1968) and Aviram and Krauss (1974) are carboxymethylation of methionyl-65 and protonation of the axially coordinated histidyl- 18. Carboxymethylation of other residues, which are less susceptible to reaction than methionyl-65, during long reaction times (Eakin et al., 1975) creates
the potential for further variation in the 13C0 chemical shift. A simple model, based on the above considerations and which is consistent with the bulk of previously reported observations on both unmodified and alkylated ferrocytochromes c, may be proposed in terms of the following sets of reactions: pH 4.5
+ H+ + X-FeII-His + X + HisH+ + CO-FeIr-H20 C O + X-FeII-His + X + CO-FeII-His
(1)
(2)
In these equations X is an unspecified axial ligand which can be a particular amino acid side chain, water, an anion, or nothing. In eq 1, X represents a methionyl residue for unmodified cytochrome. Below pH 4.5, unmodified and alkylated cytochrome c behave similarly, both with respect to the observation of single peak spectra and to the peak position as a function of pH. The apparent lack of dependence on the nature of derivatives, seen in Figure 1, suggests strongly that similar heme configurations and ligand environments are present, as might be expected if the protein is at least partially unfolded and a position on the iron(I1) is available for CO'coordination. The progressive downfield shift of the 13C0resonance with decreasing pH may then be attributed to further unfolding of the protein, which is apparently complete at pH 2. The shape and range of the shift vs. pH curve in that region implies that protonation of various side-chain carboxyl groups participates in the process through changes in the tertiary structure as pH is decreased. Spectral evidence presented by Stellwagen and Babul (1975; Babul and Stellwagen, 1972) supports conclusions that ferricytochrome c, in the presence of 1 M chloride (or greater), changes to another globular conformation upon acidification to pH 2, in which the methionine ligand is replaced by a solvent-supplied weak field ligand, but ligation of the heme by histidine-18 is not disturbed. High resolution proton N M R spectra (Gupta and Koenig, 1971) indicate loss of the resonance attributable to methyl protons of coordinated methionine-80 below pH 4 at moderate ionic strengths, and the sulfur of methionine-80 is readily alkylated at pH 3, as previously noted. Protonation of one of three histidine side-chain imidazole groups occurs below pH 2 (Cohen et al., 1974). Although Fisher et al. (1 973) have observed that porphyrincytochrome c has a compact structure similar to that of native cytochrome c, so that coordination of methionine-80 and histidine-18 is not required for protein folding, the decreased thermal stability of the former indicates that coordination contributes to the stability of the compact conformation and may be required for proper conformation of the heme crevice in the native cytochrome. The abrupt change in chemical shift of I3CO observed in alkylated cytochrome c at pH 4.5-5 is characteristic of a single protonation process and occurs at the pH where the carbonyl complex of unmodified cytochrome c begins to be detectable. Results of potentiometric titration of ferrocytochrome c, (PalCus, 1954) indicated that the propionate side chains of the heme group have pK = 5.4. More recent work (Shaw and Hartzell, 1975) suggests that the propionates, which are thought to contribute to proper configuration of the heme crevice through hydrogen bonding to a number of amino acid residues (Dickerson et al., 1971; Aviram and Schejter, 1971; Salemme et al., 1973; Tanaka et al., 1975) are protonated at pH 4.8 and 5.4. Below pH 4.8, alteration of the tertiary structure as a result of the protonation could then be responB I O C H E M I S T R Y , V O L . 1 5 , N O . 10,
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sible for C O insertion in unmodified cytochrome c and for the change in the heme environment leading to the sharp upfield shift of the 13C0 resonance in the modified cytochrome c complexes. In the pH range 5-9, the principal observable phenomena are the heterogeneity of species formed in carboxymethylation of cyanoferricytochrome c and the greater apparent stability of the CO complex species. At least three well-defined carbonyl complexes are indicated, providing heme environments designated X in eq 2. The principal downfield component may be correlated with a heme environment in which only methionyl-80 has been carboxymethylated and the principal upfield component, to one which includes a carboxymethylated methionyl-65 residue, as well. While it is possible that the carboxyl group of the carboxymethylated methionine-65 could contribute directly to the local environment of iron-bound CO, it is more likely that modification of that residue gives rise to a long range conformational change in the heme crevice. The upfield shoulder, a poorly resolved, but relatively abundant, component observable after long alkylation reaction times, can be attributed to an environment containing one or more carboxymethyl derivatives in addition to the two carboxymethylmethionyl residues. Only one observation was made above pH 9, on a solution of (ldCM)c, for the purpose of identifying the source of the upfield spectral component in the I3CO spectrum. At pH 11, hydrolysis of the carboxylate esters is relatively rapid and elimination of the peak at that pH could indicate that one or more of the glycolate ester groups is responsible for the conformation giving that component. Although a single, weak, shifted peak was indeed observed at pH 11, recovery to the more intense two-peak system upon return to lower pH suggests that a modification other than a glycolate ester product is responsible for the upfield component. It is probable that appearance of the single 13C0 peak at 205.55 ppm in the pH 11 solution is attributable to partial unfolding of the protein chain to provide a conformation with a relatively hydrophilic heme environment in which differences in alkylation of the various species have little significancewith respect to the bound I3CO spectrum. Although the heme species at high pH is shown in eq 2 with a histidine ligand, further deprotonation of the bound histidine imidazole group may occur (Peisach et al., 1973) that could appear as a change in the Fe(I1)-N field, or allow displacement of histidyl-18 by hydroxide to give a complex similar to that observed in the low pH region, as suggested by the value of the chemical shift. Alternatively, the change to the so-called basic conformation may involve hydrogen bonding of propionate to histidine-18 (Shaw and Hartzell, 1975), and consequent weakening of the Fe(I1)-N field. The I3C chemical shifts shown in Figure 1 span a range of less than 1 ppm. Comparison of a large number of spectra for a given sample, and of different samples representing the same properties and experimental conditions, leads us to believe that the reported values are reproducible to within f 0 . 0 2 ppm (0.5 Hz) and that they accurately represent the trends in chemical shift. The signal for free, dissolved I3CO was not observed in any of these samples, although in saturated aqueous solutions of CO, examined under comparable conditions, the resonance is found at 184.9 ppm (downfield from neat Me&). The chemical shifts reported for various iron carbonyls fall within a fairly narrow range and are little affected by either number or nature of other substituents. For example, a series of n-CsHsFe(CO)zX compounds, with X ranging from CN- to (C,jHs),Si, exhibits shifts of 21 1.4-216.5 ppm relative to in-
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ternal Me& (Gansow et al., 1972), while I3CO bound to a variety of hemoglobins is observed in the range 205.6-208.6 ppm (Matwiyoff et al., 1973; Vergamini et al., 1973; Moon and Richards, 1972a,b). In the latter, CO is trans to the imidazole group of the proximal histidine, in a situation comparable to that in the carboxymethylcytochrome c derivatives. Metal carbonyls have not been investigated in a wide range of solvents so that no reasonable estimate of solvent effects can be made. However, in several experiments designed to explore the possible influence of hydrogen bonding on the I3C signal, it was found that the observed 13Cspectral positions for both dicarbonylchlororhodium(1) dimer and free I3CO dissolved in benzene solution were unaffected by the presence of ca. 1 M phenol (P. J. Vergamini, unpublished data). Thus, even the small changes observed in the results reported here may reflect profound alterations in the bound CO environment or iron(I1) configuration. References Ando, K., Matsubara, H., and Okunuki, K. (19 6 9 , Proc. Jpn. Acad. 41. 79. Aviram, I., and Krauss Y. (1974), J. Biol. Chem. 249, 2575. Aviram, I., and Schejter, A. (1971), Biochim. Biophy.9.Acta 229, 113. Babul, J., and Stellwagen, E. (1972), Biochemistry 11, 1195 Cohen, J. S., Fisher, W. R., and Schechter, A. N. (1974). J . Biol. Chem. 249, 1 113. Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Samson, L., Copper, A., and Margoliash, E. (1971), J . Biol. Chem. 246, 15 1 1. Eakin, R. T., Morgan, L. O., and Matwiyoff, N. A. (1975), Biochemistry 14, 4538. Fisher, W. R., Taniuchi, H., and Anfinsen, C. B. (1973), J . Biol. Chem. 248, 3188. Gansow, 0 .A., Shexnayder, D. A., and Kimura, B. Y. (1972), J. Am, Chem. SOC.94, 3406. Gupta, R. K., and Koenig, S. H. (1971), Biochem. Biophys. Res. Commun. 45, 1134. Harbury, H. A. (1966), in Hemes and Hemoproteins, Chance, B., Estabrook, R. W., and Yonetani, T., Ed., New York, N.Y., Academic Press, p 391. Harbury, H. A., Cronin, Y. R., Fanger, M. W., Hettinger, T. P., Murphy, A. J., Myer, Y., and Vinogradov, S. N. (1969, Proc. Natl. Acad. Sci. U.S.A.54, 1658. Knapp, J. A., and Pace, C. N. (1974), Biochemistry 13. 1289. Margoliash, E., Frohwirt, N., and Wiener, E. (1959), Biochem. J . 71, 559. Matwiyoff, N . A., Vergamini, P. J., Needham, T., Gregg, C. T., Volpe, J. A., and Caughey, W. S. (1973), J . Am. Chem. SOC. 95, 443 1. Moon, R. B., and Richards, J. H. (1972a), J. Am. Chem. SOC. 94, 5093. Moon, R. B., and Richards, J. H. (1972b), Proc. Natl. Acad. Sci. U.S.A.69, 2193. PalCus, S . (1954), Acta Chem. Scand. 8, 971. Peisach, J., Blumberg, W. F., and Adler, A. (1973), Ann. N. Y. Acad. Sci. 206, 3 10. Salemme, F. R., Kraut, J., and Kamen, M. D. (1973), J . Biol. Chem. 248, 7701. Schejter, A., and Aviram, I. (1970), J. Biol. Chem. 245, 1552. Shaw, R. W., and Hartzell, C. R. (1979, Fed. Proc., Fed. Am. SOC.Exp. Biol. 34, 603; also, private communication, to be published. Stellwagen, E. (1966), Biochem. Biophys. Res. Commun. 23, 29.
PHYSICAL PROPERTIES OF CARBAMOYLATED HEMOGLOBINS
Stellwagen, E. (1968), Biochemistry 7, 2496. Stellwagen, E., and Babul, J. (1975), Biochemistry 14, 5135. Tanaka, N., Yamane, T., Tsukihara, T., Ashida, T., and Kakudo, M. (1975), J . Biochem. (Tokyo) 77, 147.
Tsai, H. J., and Williams, G. R. (1965), Can. J. Biochem. 43, 1409. Vergamini, P. J., Matwiyoff, N. A., Wohl, R. C., and Bradley, T. (1973), Biochem. Biophys. Res. Commun. 55, 453.
Carbamoylated Hemoglobins A and S: Physical Properties? Robley C. Williams, Jr.,* and Helen Kim
ABSTRACT: Dimer-tetramer association constants (K2.4) of derivatives of CO-hemoglobins A and S specifically carbamoylated at the “2-terminal valine residues were measured. Reactivities of the p-93 sulfhydryls of the hemoglobin A derivatives were also investigated. As compared with the association constants of the parent molecules, the values of K2,4 of both hemoglobin types are raised by carbamoylation of the a-chain NH2 terminus, lowered by carbamoylation of the pchain NH2 terminus, and raised by carbamoylation of both termini. The apparent second-order rate constant for reaction of p-mercuribenzoate (PMB) with the 0-93 sulfhydryls is, however, unchanged by carbamoylation. These two observations are interpreted to indicate that in the liganded molecule
structural changes are produced at the interface between dimers but not in the region of the 6-93 sulfhydryls. From the combination of the K2.4 measurements with ligand-binding data for the same derivatives (Kilmartin, J. V., et al. (1973), J. Biol. Chem. 248, 7039; Nigen, A. M., et al. (1974), J. Biol. Chem. 249, 661 1) the carbamoylation-induced changes in the dimer-tetramer association constants of the unliganded derivatives were estimated to be of magnitude equal to or smaller than those in K2,4. It is concluded that much of the change in oxygen affinity that occurs upon carbamoylation of hemoglobins A and S can be accounted for without invoking extensive structural changes in the unliganded molecule.
H e m o g l o b i n s specifically carbamoylated at the NHz-terminal valine residues of the a-or &chains have been useful in the elucidation of a number of functional properties of the molecule. The carbamoyl group has been employed as a modifier in studies of the alkaline Bohr effect and of the binding of C 0 2 and 2,3-diphosphoglycerate to hemoglobin (Kilmartin and Rossi-Bernardi, 1971; Kilmartin et al., 1973; Kilmartin, 1974). The rates of carbamoylation of the “2terminal groups of hemoglobin have also been exploited (Garner et al., 1975) to measure the pK‘s of those groups. Additional interest in the properties of these derivatives has been stimulated by the finding of Cerami and Manning (1971) that carbamoylation of hemoglobin S (HbS’) in the erythrocytes of sickle-cell homozygotes leads to an inhibition of sickling, and that this effect is directly related to carbamoylation of the NH2 termini. This inhibition apparently results partly from an elevation of oxygen affinity (de Furia et al., 1972;May et al., 1972), partly from changes in the conformation of the partially or fully deoxygenated HbS molecule which are reflected in an increase in the minimum concentration at which the protein will gel (Williams, 1973; Nigen et al., 1974), and partly from other less well understood factors (Wagner et al., 1975). This paper reports a study of the dimer-tetramer association behavior and sulfhydryl reactivity of the carbamoyl derivatives of HbA and HbS. The results are interpreted tentatively in
terms of conformational differences which may be induced by carbamoylation.
From the Department of Biology, Yale University, New Haven, Connecticut 06520. Received October 17, 1975. This research was supported by Grant H L 12901 of the National Institutes of Health. Abbreviations used: PMB, p-mercuribenzoate; HbS, hemoglobin S ; HbA, hemoglobin A; oxy-Hb, oxyhemoglobin; Tris, tris(hydr0xymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid.
Materials and Methods Reagent grade chemicals were used throughout, with the exceptions of Tris, which was “Ultra Pure” (Mann), and KNCO, which was recrystallized from the best available grade (initially 97% KNCO). Preparation of Specifically Carbamoylated Hemoglobins. HbA, free of minor hemoglobin components and of organic phosphates, was prepared (as oxy-Hb) by the chromatographic method of Williams and Tsay (1973). HbS was prepared from the erythrocytes of an individual known to be homozygous for the sickle-cell gene by a modification of that method in which chromatography was performed at pH 7.8 instead of the pH 7.6 employed with HbA. Specifically carbamoylated hemoglobins were prepared and separated chromatographically (as CO-Hb) exactly as described by Williams et al. (1975). The three modified hemoglobins obtained are apC, aCp,and acpC, where the superscript c implies the presence of a carbamoylated NH2 terminus. Measurement of Association Constants. Dimer-tetramer association of the CO-hemoglobins was measured by equilibrium centrifugation in a Beckman Model E ultracentrifuge. The instrument employs a modified absorption scanner that operates in conjunction with an on-line computer (Williams, 1976). The scanner was calibrated both for linearity and absolute absorbance against a Gilford spectrophotometer at 4 19 nm. Absorbance measurements were converted to molarity of heme by means of this calibration and the extinction coefficient (1.91 X lo5 M-l) of CO-Hb at 419 nm (Rossi-Fanelli et al., 1959). Centrifuge cells of 30-mm optical path of the type described by Ansevin et al. (1 970) were employed, together with BIOCHEMISTRY, VOL.
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Carbon- 13 Nuclear Magnetic Resonance of Heme Carbonyls. Cytochrome c and Carboxymethyl Derivatives of Cytochrome c t L. 0. Morgan,*.i R. T. Eakin, P. J. Vergamini, and N. A. Matwiyoff
ABSTRACT: Carbonyl complexes of horse cytochrome c and various carboxymethylated derivatives have been examined using I3C N M R (carbon- 13 nuclear magnetic resonance) spectroscopy. The multiplicity and chemical shift of the I3CO resonance were found to be functions of pH and the extent of alkylation. Correlations have been made among prominent
c
ytochrome c is a low molecular weight (1 2 400 daltons) heme protein whose tertiary structure has been highly conserved throughout evolution. The structural similarity of the cytochromes c among a wide variety of plant and animal species is indicative of the critical importance of the electronic environment of the heme group in the functional role of this protein as a biological electron carrier. Thus, the determination of the electronic environment and protein configuration in the vicinity of the heme of cytochrome c is of particular importance in understanding the mechanism by which this protein transfers electrons in biological oxidation processes. One of the classical methods of probing the electronic environment of heme in heme proteins is through the formation of complexes with small ligands such as carbon monoxide, molecular oxygen, or nitrous oxide. Visible and infrared spectroscopy of such complexes have given some indication of the structural and electronic nature of the heme environment, and recently formation of carbonyl complexes with 13C0has provided a more sensitive probe via 13C N M R (carbon-13 nuclear magnetic resonance) spectroscopy. This method has shown differences, for example, among the heme environments of several heme protein-CO complexes, including sperm whale myoglobin (Moon and Richards, 1972a) and the CY and 0 chains of various species of hemoglobins (Matwiyoff et al., 1973; Vergamini et al., 1973; Moon and Richards, 1972a,b). In the native state of cytochrome c, the histidyl-18 and the methionyl-80 residues of the protein serve as axial ligands for the heme group and prevent formation of carbonyl complexes. However, procedures which remove one or both of these axial ligands, such as carboxymethylation in the presence of cyanide (Schejter and Aviram, 1970), lowering the pH (Cohen et al., 1974), or denaturing in urea or guanidine hydrochloride (Knapp and Pace, 1974), allow the formation of carbonyl derivatives. We have investigated the carbonyl complexes of some of these derivatives using 13CN M R spectroscopy and, in the From the Department of Chemistry, The University of Texas at Austin (L.O.M.and R.T.E.),Austin, Texas 78712, and The Los Alamos Scientific Laboratory (P.J.V. and N.A.M.), University of California, Los Alamos, New Mexico 87545. Received November 18. 1975. This work was performed under the auspices of the United States Energy Research and Development Administration and was supported in part by the Robert A. Welch Foundation, Houston, Texas. University of Texas at Austin, University Research Institute grantin-aid.
*
features of the chemical shift titration curves and changes in the environment of the heme. A simple model compatible with the bulk of previous observationshas been suggested to account for the several carbonyl resonance peaks and the complex behavior of the chemical shift with changes in pH.
results presented here, describe pH-dependent conformational transitions and heme environment heterogeneity. Materials and Methods Sources of Materials. All alkylated derivatives of cytochrome c were prepared from horse heart cytochrome c (Sigma Chemical Company, type VI). Natural abundance carbon (1.1 atoms %) bromoacetic acid was obtained from Eastman Kodak. [2-13C]Br~m~acetic acid used in some of the carboxymethylation reactions and [ 12C]urea used for denaturing cytochrome c were a gift from Donald Ott and Vernon Kerr of the Los Alamos Scientific Laboratory. Enriched I3CO (90+ atoms %) was a gift from B. B. McInteer of the Los Alamos Scientific Laboratory. Preparation of Carboxymethylcytochrome c. Carboxymethyl derivatives of cytochrome c were prepared in several different ways, as previously reported (Eakin et al., 1975). In that work, estimates of carboxymethyl product formation in the various types of amino acid residues from alkylation with [2-13C]bromoacetate were obtained from 13C N M R spectra of the modified proteins. Major features of the alkylation processes were found to conform with those obtained in other laboratories, although evidence for glycolate ester formation and more extensive lysyl modification appeared in the 13C NMR spectroscopic analyses. Three classes of carboxymethyl derivatives used in this work may be described as follows: (1) Dicarboxymethylmethionylcytochromec [(CmMet)2c;' Ando et al., 1965; Tsai and Williams, 1965; Babul and Stellwagen, 1972)] was formed from acid denatured cytochrome c at pH 3. Both methionyl residues were alkylated and essentially no other residues were affected. (2) Carboxymethylcytochrome c from a 6-day alkylation reaction time [(6dCm)c; Harbury et al., 1965; Stellwagen, 1968; Schejter apd Aviram, 1970)] was formed at pH 7 from cyanoferricytochrome c in 0.1 M CN-. This preparation contained 2 alkylated methionyl residues, 1 alkylated histidyl residue, 2 alkylated lysyl residues, and 2.6 glycolate ester products. (3) Carboxymethylcytochrome c from a 1-day alkylation reaction time [(ldCm)c] was formed at pH 7 from cyanoferricytochrome c in 0.1 M CN-. I Abbreviations used: (CmMet)Zc, dicarboxymethylmethionylcytochrome c; (6dCm)c, carboxymethylcytochromec from a 6-day alkylation reaction time; (ldCm)c, carboxymethylcytochrome c from a I-day alkylation reaction time.
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t
f
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1
I
1
1
1
1
I
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3
4
5
6
7
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FIGURE 1: Chemical shift titration curves for I3COcomplexes of cytochrome c derivatives; parts per million downfield from Me&i as a function of solution pH. (a) Unmodified cytochrome c; (b) (1dCm)c; (c) (6dCm)c; (d) (CmMet)zc.
The alkylation was less extensive than in the analogous 6-day reaction time preparation, amounting to the formation of 1.3 carboxymethylmethionyl residues, ca. 0.6 carboxymethylhistidyl residues, ca. 0.8 carboxymethyllysyl residues, and 0.5 glycolate ester products. On the basis of previous kinetic results (Stellwagen, 1966, 1968), we estimate that 0.9 methionyl-80 and 0.4 methionyl-65 residues were carboxymethylated, to give the observed total of 1.3. In both (1dCm)cl and (6dCm)c, dicarboxymethylation of histidyl and lysyl residues was observed and the estimated amounts are included in the specified totals. The I3C-labeled methylene group magnetic resonance peaks for monocarboxymethyllysyl and monocarboxymethylhistidyl residues could not be resolved so that the relative abundances of those modified residues listed above represent an estimate based on previous observations (Harbury, 1966; Stellwagen, 1966, 1968). Preparation of Carbonyl Derivatives of Carboxymethylcytochrome c. Carbonyl derivatives of the various preparations of carboxymethylcytochrome c were obtained by reduction of aqueous solutions with sodium dithionite. Without removing the dithionite, the solutions were saturated with I3CO from slow bubbling at atmospheric pressure for at least 10 min. After obtaining I3CO spectra on some solutions, a chemical shift titration was performed by sequential adjustment of pH with phosphoric acid, hydrochloric acid, or concentrated potassium hydroxide solution, treatment with dithionite, and resaturation with 13C0. N M R Spectroscopy. Fourier transform 3 C 0 NMR spectra were obtained at 20 OC as described previously (Eakin et al., 1975) over a 200-Hz region using 1024 data points in the time domain, with a total sampling time of 2.56 s. Although T I values for I3CO bound to iron(I1) in hemoglobins are 1.1- 1.2 s, corresponding values of T2 are very short and observed lines are not appreciably saturated. Chemical shift values were calculated in parts per million downfield relative to neat tet-
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FIGURE 2: Carbonyl resonance spectra of I3CO complexes of cytochrome 5; (b) (ldCm)c, pH 7; (c) (6dCm)c. pH
c derivatives. (a) (ldCm)c, pH
I.
ramethylsilane ( M e S i ) measured under similar conditions. Spectra were analyzed by least-squares line-shape analysis of the full transformed data set, utilizing a Fortran-based computer program which resolves complex line shapes into Lorentzian components. Chemical shifts of the peak centers and relative peak areas, along with their standard deviations, are calculated from the resolved curves as parts of the program. Results Chemical shifts of carbonyl resonances were measured over the pH range 2-9 for Fe(II)-I3CO complexes of unmodified cytochrome c (Figure la), (1dCm)c (Figure lb), (6dCm)c (Figure IC), and (CmMet)2c (Figure Id). Reduction of unmodified cytochrome c in aqueous solution with sodium dithionite, followed by saturation of the solution with 13C0did not produce a detectable carbonyl complex above pH 5. At pH 4.73 a weak resonance peak appeared at 205.40 ppm. Progressive lowering of the pH resulted in the chemical shift titration curve shown in Figure la, with peak intensity reaching a maximum in the pH range 3-4. No significant differences were observed between shifts observed in samples adjusted with phosphoric acid and with hydrochloric acid. Similar treatment of the three different carboxymethylcytochrome preparations yielded complexes exhibiting carbonyl resonances over the entire experimental range, pH 2-9. Representative spectra are shown in Figure 2. In [13CO]-(ldCm)c two prominent components appeared (Figure 2b), with peak positions indicated as separate branches of the titration curve (Figure lb) above pH 5. At lower pH values a single resonance peak was observed (Figure 2a) for which the spectral position and pH dependence were essentially the same as those found for [13CO]-(6dCm)c (Figure IC) and [13CO]-(CmMet)2c (Figure Id). A single resonance was observed at 205.55 ppm when the pH was increased to 11 .O. Subsequent adjustment of the solution pH to 7.1 caused a reappearance of the two-peak system. The carbonyl complex of (6dCm)c produced a spectrum comprising a single principal peak, although a small
N M R OF HEME CARBONYLS
shoulder, 0.8 ppm upfield from the peak maximum, was discernible (Figure 2c) at all except the lowest pH values, where it could easily have been masked by line broadening, or lost in the noise. In general, the behavior of the shoulder followed that of the main peak and maintained a constant relative amplitude over the pH range, where it was observable. For intermediate alkylation periods, with other variables held constant, the derivatized cytochromes gave carbonyl complexes displaying two-component spectra in the carbonyl region. Chemical shifts of the two components were the same as those in (1dCm)c and relative intensities varied in a linear fashion between the 1- and 6-day extremes. At reaction times of 1, 2.5, and 6 days, the fractional intensities of the upfield peak were 0.35, 0.55, and 1.0, respectively. In the first case, the relative intensity of the upfield peak was observed to increase to ca. 0.5 upon decrease of pH to 5.3. On the basis of total peak integrals and observed signalto-noise ratios obtained under comparable spectrometric conditions, it appeared that for the carboxymethyl derivatives, C O coordination to the heme group was optimal in the pH range 5-7. Because of variations in both protein concentration and ionic strength in the usual titration procedure, such a comparison is qualitative, at best. An attempt was made to improve the accuracy as follows. A sample of (1dCm)c was desalted on a Sephadex G-25 column, freeze-dried, and dissolved at 100 mg/ml concentration in four potassium phosphate solutions of differing pH, but constant phosphate concentration (1 .OO M). After dithionite reduction and saturation with I3CO gas, final pH values were 4.47,5.00,5.47, and 8.30. With constant spectrometer settings and with the same numbers of rf pulses, total integrated intensities were found to be 0.64,0.9 1, 1.OO, and 0.78, respectively, normalized to the peak intensity of the pH 5.47 solution. Discussion The experimental results clearly establish that the chemical shift of the I3CO resonance in carbonyl complexes of carboxymethylcytochrome c is sensitive to both pH and extent of protein alkylation. Although the titration curves of Figure 1 appear complex, the data can easily be correlated with reasonable changes in the electronic environment at the heme binding site due to variation in pH or alkylation of amino acid side chains at critical positions. At extremes of pH the protein chain is likely to be unfolded to an extent that the hydrophobic cleft is replaced by a more hydrophilic environment dominated by solvent water. In such cases the extent of alkylation of the protein would not be expected to have a significant effect on the chemical shift position of the heme bound carbonyl resonance, although it might affect details of the protein conformation. I n intermediate pH regions where the tertiary protein structure is still in its compact globular state, carbonyl complexes will be observed only when the methionyl-80 residue has been displaced from its axial coordination position, as by carboxymethylation. The modification of other proximal amino acid side chains could be expected to influence the position of the carbonyl resonance in this pH range where the protein chain can contribute substantially to the electronic environment of the heme group. Two such additional modifications which can be anticipated from previous observations by Stellwagen (1968) and Aviram and Krauss (1974) are carboxymethylation of methionyl-65 and protonation of the axially coordinated histidyl- 18. Carboxymethylation of other residues, which are less susceptible to reaction than methionyl-65, during long reaction times (Eakin et al., 1975) creates
the potential for further variation in the 13C0 chemical shift. A simple model, based on the above considerations and which is consistent with the bulk of previously reported observations on both unmodified and alkylated ferrocytochromes c, may be proposed in terms of the following sets of reactions: pH 4.5
+ H+ + X-FeII-His + X + HisH+ + CO-FeIr-H20 C O + X-FeII-His + X + CO-FeII-His
(1)
(2)
In these equations X is an unspecified axial ligand which can be a particular amino acid side chain, water, an anion, or nothing. In eq 1, X represents a methionyl residue for unmodified cytochrome. Below pH 4.5, unmodified and alkylated cytochrome c behave similarly, both with respect to the observation of single peak spectra and to the peak position as a function of pH. The apparent lack of dependence on the nature of derivatives, seen in Figure 1, suggests strongly that similar heme configurations and ligand environments are present, as might be expected if the protein is at least partially unfolded and a position on the iron(I1) is available for CO'coordination. The progressive downfield shift of the 13C0resonance with decreasing pH may then be attributed to further unfolding of the protein, which is apparently complete at pH 2. The shape and range of the shift vs. pH curve in that region implies that protonation of various side-chain carboxyl groups participates in the process through changes in the tertiary structure as pH is decreased. Spectral evidence presented by Stellwagen and Babul (1975; Babul and Stellwagen, 1972) supports conclusions that ferricytochrome c, in the presence of 1 M chloride (or greater), changes to another globular conformation upon acidification to pH 2, in which the methionine ligand is replaced by a solvent-supplied weak field ligand, but ligation of the heme by histidine-18 is not disturbed. High resolution proton N M R spectra (Gupta and Koenig, 1971) indicate loss of the resonance attributable to methyl protons of coordinated methionine-80 below pH 4 at moderate ionic strengths, and the sulfur of methionine-80 is readily alkylated at pH 3, as previously noted. Protonation of one of three histidine side-chain imidazole groups occurs below pH 2 (Cohen et al., 1974). Although Fisher et al. (1 973) have observed that porphyrincytochrome c has a compact structure similar to that of native cytochrome c, so that coordination of methionine-80 and histidine-18 is not required for protein folding, the decreased thermal stability of the former indicates that coordination contributes to the stability of the compact conformation and may be required for proper conformation of the heme crevice in the native cytochrome. The abrupt change in chemical shift of I3CO observed in alkylated cytochrome c at pH 4.5-5 is characteristic of a single protonation process and occurs at the pH where the carbonyl complex of unmodified cytochrome c begins to be detectable. Results of potentiometric titration of ferrocytochrome c, (PalCus, 1954) indicated that the propionate side chains of the heme group have pK = 5.4. More recent work (Shaw and Hartzell, 1975) suggests that the propionates, which are thought to contribute to proper configuration of the heme crevice through hydrogen bonding to a number of amino acid residues (Dickerson et al., 1971; Aviram and Schejter, 1971; Salemme et al., 1973; Tanaka et al., 1975) are protonated at pH 4.8 and 5.4. Below pH 4.8, alteration of the tertiary structure as a result of the protonation could then be responB I O C H E M I S T R Y , V O L . 1 5 , N O . 10,
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sible for C O insertion in unmodified cytochrome c and for the change in the heme environment leading to the sharp upfield shift of the 13C0 resonance in the modified cytochrome c complexes. In the pH range 5-9, the principal observable phenomena are the heterogeneity of species formed in carboxymethylation of cyanoferricytochrome c and the greater apparent stability of the CO complex species. At least three well-defined carbonyl complexes are indicated, providing heme environments designated X in eq 2. The principal downfield component may be correlated with a heme environment in which only methionyl-80 has been carboxymethylated and the principal upfield component, to one which includes a carboxymethylated methionyl-65 residue, as well. While it is possible that the carboxyl group of the carboxymethylated methionine-65 could contribute directly to the local environment of iron-bound CO, it is more likely that modification of that residue gives rise to a long range conformational change in the heme crevice. The upfield shoulder, a poorly resolved, but relatively abundant, component observable after long alkylation reaction times, can be attributed to an environment containing one or more carboxymethyl derivatives in addition to the two carboxymethylmethionyl residues. Only one observation was made above pH 9, on a solution of (ldCM)c, for the purpose of identifying the source of the upfield spectral component in the I3CO spectrum. At pH 11, hydrolysis of the carboxylate esters is relatively rapid and elimination of the peak at that pH could indicate that one or more of the glycolate ester groups is responsible for the conformation giving that component. Although a single, weak, shifted peak was indeed observed at pH 11, recovery to the more intense two-peak system upon return to lower pH suggests that a modification other than a glycolate ester product is responsible for the upfield component. It is probable that appearance of the single 13C0 peak at 205.55 ppm in the pH 11 solution is attributable to partial unfolding of the protein chain to provide a conformation with a relatively hydrophilic heme environment in which differences in alkylation of the various species have little significancewith respect to the bound I3CO spectrum. Although the heme species at high pH is shown in eq 2 with a histidine ligand, further deprotonation of the bound histidine imidazole group may occur (Peisach et al., 1973) that could appear as a change in the Fe(I1)-N field, or allow displacement of histidyl-18 by hydroxide to give a complex similar to that observed in the low pH region, as suggested by the value of the chemical shift. Alternatively, the change to the so-called basic conformation may involve hydrogen bonding of propionate to histidine-18 (Shaw and Hartzell, 1975), and consequent weakening of the Fe(I1)-N field. The I3C chemical shifts shown in Figure 1 span a range of less than 1 ppm. Comparison of a large number of spectra for a given sample, and of different samples representing the same properties and experimental conditions, leads us to believe that the reported values are reproducible to within f 0 . 0 2 ppm (0.5 Hz) and that they accurately represent the trends in chemical shift. The signal for free, dissolved I3CO was not observed in any of these samples, although in saturated aqueous solutions of CO, examined under comparable conditions, the resonance is found at 184.9 ppm (downfield from neat Me&). The chemical shifts reported for various iron carbonyls fall within a fairly narrow range and are little affected by either number or nature of other substituents. For example, a series of n-CsHsFe(CO)zX compounds, with X ranging from CN- to (C,jHs),Si, exhibits shifts of 21 1.4-216.5 ppm relative to in-
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ternal Me& (Gansow et al., 1972), while I3CO bound to a variety of hemoglobins is observed in the range 205.6-208.6 ppm (Matwiyoff et al., 1973; Vergamini et al., 1973; Moon and Richards, 1972a,b). In the latter, CO is trans to the imidazole group of the proximal histidine, in a situation comparable to that in the carboxymethylcytochrome c derivatives. Metal carbonyls have not been investigated in a wide range of solvents so that no reasonable estimate of solvent effects can be made. However, in several experiments designed to explore the possible influence of hydrogen bonding on the I3C signal, it was found that the observed 13Cspectral positions for both dicarbonylchlororhodium(1) dimer and free I3CO dissolved in benzene solution were unaffected by the presence of ca. 1 M phenol (P. J. Vergamini, unpublished data). Thus, even the small changes observed in the results reported here may reflect profound alterations in the bound CO environment or iron(I1) configuration. References Ando, K., Matsubara, H., and Okunuki, K. (19 6 9 , Proc. Jpn. Acad. 41. 79. Aviram, I., and Krauss Y. (1974), J. Biol. Chem. 249, 2575. Aviram, I., and Schejter, A. (1971), Biochim. Biophy.9.Acta 229, 113. Babul, J., and Stellwagen, E. (1972), Biochemistry 11, 1195 Cohen, J. S., Fisher, W. R., and Schechter, A. N. (1974). J . Biol. Chem. 249, 1 113. Dickerson, R. E., Takano, T., Eisenberg, D., Kallai, 0. B., Samson, L., Copper, A., and Margoliash, E. (1971), J . Biol. Chem. 246, 15 1 1. Eakin, R. T., Morgan, L. O., and Matwiyoff, N. A. (1975), Biochemistry 14, 4538. Fisher, W. R., Taniuchi, H., and Anfinsen, C. B. (1973), J . Biol. Chem. 248, 3188. Gansow, 0 .A., Shexnayder, D. A., and Kimura, B. Y. (1972), J. Am, Chem. SOC.94, 3406. Gupta, R. K., and Koenig, S. H. (1971), Biochem. Biophys. Res. Commun. 45, 1134. Harbury, H. A. (1966), in Hemes and Hemoproteins, Chance, B., Estabrook, R. W., and Yonetani, T., Ed., New York, N.Y., Academic Press, p 391. Harbury, H. A., Cronin, Y. R., Fanger, M. W., Hettinger, T. P., Murphy, A. J., Myer, Y., and Vinogradov, S. N. (1969, Proc. Natl. Acad. Sci. U.S.A.54, 1658. Knapp, J. A., and Pace, C. N. (1974), Biochemistry 13. 1289. Margoliash, E., Frohwirt, N., and Wiener, E. (1959), Biochem. J . 71, 559. Matwiyoff, N . A., Vergamini, P. J., Needham, T., Gregg, C. T., Volpe, J. A., and Caughey, W. S. (1973), J . Am. Chem. SOC. 95, 443 1. Moon, R. B., and Richards, J. H. (1972a), J. Am. Chem. SOC. 94, 5093. Moon, R. B., and Richards, J. H. (1972b), Proc. Natl. Acad. Sci. U.S.A.69, 2193. PalCus, S . (1954), Acta Chem. Scand. 8, 971. Peisach, J., Blumberg, W. F., and Adler, A. (1973), Ann. N. Y. Acad. Sci. 206, 3 10. Salemme, F. R., Kraut, J., and Kamen, M. D. (1973), J . Biol. Chem. 248, 7701. Schejter, A., and Aviram, I. (1970), J. Biol. Chem. 245, 1552. Shaw, R. W., and Hartzell, C. R. (1979, Fed. Proc., Fed. Am. SOC.Exp. Biol. 34, 603; also, private communication, to be published. Stellwagen, E. (1966), Biochem. Biophys. Res. Commun. 23, 29.
PHYSICAL PROPERTIES OF CARBAMOYLATED HEMOGLOBINS
Stellwagen, E. (1968), Biochemistry 7, 2496. Stellwagen, E., and Babul, J. (1975), Biochemistry 14, 5135. Tanaka, N., Yamane, T., Tsukihara, T., Ashida, T., and Kakudo, M. (1975), J . Biochem. (Tokyo) 77, 147.
Tsai, H. J., and Williams, G. R. (1965), Can. J. Biochem. 43, 1409. Vergamini, P. J., Matwiyoff, N. A., Wohl, R. C., and Bradley, T. (1973), Biochem. Biophys. Res. Commun. 55, 453.
Carbamoylated Hemoglobins A and S: Physical Properties? Robley C. Williams, Jr.,* and Helen Kim
ABSTRACT: Dimer-tetramer association constants (K2.4) of derivatives of CO-hemoglobins A and S specifically carbamoylated at the “2-terminal valine residues were measured. Reactivities of the p-93 sulfhydryls of the hemoglobin A derivatives were also investigated. As compared with the association constants of the parent molecules, the values of K2,4 of both hemoglobin types are raised by carbamoylation of the a-chain NH2 terminus, lowered by carbamoylation of the pchain NH2 terminus, and raised by carbamoylation of both termini. The apparent second-order rate constant for reaction of p-mercuribenzoate (PMB) with the 0-93 sulfhydryls is, however, unchanged by carbamoylation. These two observations are interpreted to indicate that in the liganded molecule
structural changes are produced at the interface between dimers but not in the region of the 6-93 sulfhydryls. From the combination of the K2.4 measurements with ligand-binding data for the same derivatives (Kilmartin, J. V., et al. (1973), J. Biol. Chem. 248, 7039; Nigen, A. M., et al. (1974), J. Biol. Chem. 249, 661 1) the carbamoylation-induced changes in the dimer-tetramer association constants of the unliganded derivatives were estimated to be of magnitude equal to or smaller than those in K2,4. It is concluded that much of the change in oxygen affinity that occurs upon carbamoylation of hemoglobins A and S can be accounted for without invoking extensive structural changes in the unliganded molecule.
H e m o g l o b i n s specifically carbamoylated at the NHz-terminal valine residues of the a-or &chains have been useful in the elucidation of a number of functional properties of the molecule. The carbamoyl group has been employed as a modifier in studies of the alkaline Bohr effect and of the binding of C 0 2 and 2,3-diphosphoglycerate to hemoglobin (Kilmartin and Rossi-Bernardi, 1971; Kilmartin et al., 1973; Kilmartin, 1974). The rates of carbamoylation of the “2terminal groups of hemoglobin have also been exploited (Garner et al., 1975) to measure the pK‘s of those groups. Additional interest in the properties of these derivatives has been stimulated by the finding of Cerami and Manning (1971) that carbamoylation of hemoglobin S (HbS’) in the erythrocytes of sickle-cell homozygotes leads to an inhibition of sickling, and that this effect is directly related to carbamoylation of the NH2 termini. This inhibition apparently results partly from an elevation of oxygen affinity (de Furia et al., 1972;May et al., 1972), partly from changes in the conformation of the partially or fully deoxygenated HbS molecule which are reflected in an increase in the minimum concentration at which the protein will gel (Williams, 1973; Nigen et al., 1974), and partly from other less well understood factors (Wagner et al., 1975). This paper reports a study of the dimer-tetramer association behavior and sulfhydryl reactivity of the carbamoyl derivatives of HbA and HbS. The results are interpreted tentatively in
terms of conformational differences which may be induced by carbamoylation.
From the Department of Biology, Yale University, New Haven, Connecticut 06520. Received October 17, 1975. This research was supported by Grant H L 12901 of the National Institutes of Health. Abbreviations used: PMB, p-mercuribenzoate; HbS, hemoglobin S ; HbA, hemoglobin A; oxy-Hb, oxyhemoglobin; Tris, tris(hydr0xymethy1)aminomethane; EDTA, ethylenediaminetetraacetic acid.
Materials and Methods Reagent grade chemicals were used throughout, with the exceptions of Tris, which was “Ultra Pure” (Mann), and KNCO, which was recrystallized from the best available grade (initially 97% KNCO). Preparation of Specifically Carbamoylated Hemoglobins. HbA, free of minor hemoglobin components and of organic phosphates, was prepared (as oxy-Hb) by the chromatographic method of Williams and Tsay (1973). HbS was prepared from the erythrocytes of an individual known to be homozygous for the sickle-cell gene by a modification of that method in which chromatography was performed at pH 7.8 instead of the pH 7.6 employed with HbA. Specifically carbamoylated hemoglobins were prepared and separated chromatographically (as CO-Hb) exactly as described by Williams et al. (1975). The three modified hemoglobins obtained are apC, aCp,and acpC, where the superscript c implies the presence of a carbamoylated NH2 terminus. Measurement of Association Constants. Dimer-tetramer association of the CO-hemoglobins was measured by equilibrium centrifugation in a Beckman Model E ultracentrifuge. The instrument employs a modified absorption scanner that operates in conjunction with an on-line computer (Williams, 1976). The scanner was calibrated both for linearity and absolute absorbance against a Gilford spectrophotometer at 4 19 nm. Absorbance measurements were converted to molarity of heme by means of this calibration and the extinction coefficient (1.91 X lo5 M-l) of CO-Hb at 419 nm (Rossi-Fanelli et al., 1959). Centrifuge cells of 30-mm optical path of the type described by Ansevin et al. (1 970) were employed, together with BIOCHEMISTRY, VOL.
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