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Biochemistry 1994,33, 13132-13139
Modeling the Hemoglobin Switchpoint with Cyanomet Valency Hybrids: Raman Spectroscopic Probes of Tertiary and Quaternary Structure+ Ishita Mukerji and Thomas G. Spiro’ Department of Chemistry, Princeton University, Princeton, New Jersey 08544 Received May 6, 1994; Revised Manuscript Received August 15, 1994’
/3 chains have been investigated by resonance Raman (RR) spectroscopy, using ultraviolet (230 nm) and visible (441.6 nm) excitation. For the C O adducts, the UVRR spectra are identical with that of native HbCO, showing the tyrosine and tryptophan signals to be insensitive to ligand substitution within the R state. In the absence of CO, the doubly ligated hybrids show differences in the UVRR spectra, relative to the C O adducts, which are the superposition of two difference spectra: (1) the T-R difference spectrum obtained by subtracting the spectrum of HbCO from that of deoxyHb and (2) a perturbed R state spectrum, characteristic of deligated chains within the R state. These spectra arise from alterations, respectively, in the quaternary contacts of interface aromatic residues and in the tertiary contacts of interior aromatic residues. From the amplitudes of the difference spectra, the T state population was determined to be 30% for (aFe11,!3Fc111CN)2 and 43% for (aFe11rCN/3Fe11)2, in good agreement with the kinetic analysis of Cassoly and Gibson [Cassoly, R., & Gibson, Q. H. (1972) J. Biol. Chem. 247,73321. Addition of inositol hexaphosphate (IHP) increased the T state population, but only by a modest amount, to 40 and 53%, respectively, in contrast to the frequent assumption that the T state conversion is quantitative in the presence of IHP. Since current understanding of the quaternary state dependence of the Fe-histidine stretching frequency is based on that assumption, the R R band envelope for this vibration was reexamined. For ( c Y ~ ~ ~ ~the/ band ~ ~ is~ readily ~ ~ ~resolvable C N ) ~ into two components, whose areas yield the same T / R population ratios as do the UVRR difference spectra, in the presence and in the absence of IHP. The resolved components yield 223 and 210 cm-1 as the R and T state frequencies; they both shift significantly upon I H P binding, to 219 and 203 cm-l, revealing an I H P effect on tertiary as well as quaternary structure. For (aFe111CN/3Fe11)2, the components are not independently resolvable, but fixing their areas to correspond to the UVRR-derived T / R ratios yielded R and T state frequencies of 222 and 21 3 cm-l, with no perceptible shift upon I H P binding. The T / R ratios are much lower than those derived from oxygenation curves for CN- adducts of partially oxidized H b [Marden, M. C., Kiger, L., Kister, J., Bohn, B., & Poyart, C. (1991) Biophys. J. 60,7701. This apparent discrepancy is ascribed to a preponderance of asymmetric hybrids (,,Fe11p,Fe111,2Fer11CNp2Fe111CN), which Ackers et al. [Ackers, G. K., Doyle, M. L., Myers, D., & Daugherty, M. A. (1992) Science 255, 541 have shown to be thermodynamically more stable than the symmetric hybrids. The R state perturbation for unligated chains which is revealed in the UVRR spectra is believed to involve collapse of the E helix toward the heme and weakening of a tryptophan hydrogen bond between the E and A helices. This perturbation is suggested to account for the phenomenon of quaternary enhancement, an R state augmentation of ligand affinity for unligated chains. ABSTRACT: Hybrid hemoglobins with cyanomet hemes in the a or in the
The molecular mechanism of hemoglobin cooperativity is of great importance tocurrent thinking about protein dynamics and allostery. The classical two-state model of Monod et al. (1965) captures the main features of Hbl cooperativity, although additional states are clearly available to Hb under various conditions (Ackers et al., 1992; Smith et al., 1991; Silva et al., 1992). The crystal structures of deoxyHb, and of various ligated Hb’s (Fermi & Perutz, 1977; Ladner et al., 1977; Baldwin & Chothia, 1979; Shanaan, 1983; Fermi et al., 1984), are widely accepted to represent the low- and highaffinity quaternary states, T and R. The reaction coordinate between the R and T states remains largely undefined, however, and is the object of much current research. Because of ?This work was supported by NIH Grant GM 25158. I.M. was supported by NRSA fellowship (GM14324). * Author to whom correspondence should be addressed. e Abstract published in Advunce ACS Abstracts, October 1 , 1994. Abbreviations: DPG, 2,3-diphosphoglycerate; CNmetHb, cyanomethemoglobin; FmetHb, fluoromethemoglobin; Hb, hemoglobin; IHP, inositol hexaphosphate; UVRR, ultraviolet resonance Raman spectrosCOPY; (c~+CN@)~, (aFc111CN@Fc11)2; (a@+CN)*,(aFc11@Fc111CN)2.
0006-2960/94/0433-13 132$04.50/0
cooperativity, intermediate states are sparsely populated at equilibrium and must be studied with kinetic techniques (Sawicki & Gibson, 1976, 1979; Hofrichter et al., 1983) or by chemical modification of the protein. A particularly useful modification is to oxidize two of the four hemes to the “met” form and bind the Fe3+ ions with CN- ligands, producing “valency hybrids”. Hb molecules which contain two cyanomet hemes resemble the diligated state of native Hb, which is difficult to isolate because of ligand redistribution and cooperativity. Symmetrical valency hybrids, in which the cyanomet hemes are located exclusively in the CY or in the chains, are readily produced by selective chemical treatment of separated chains, which are then recombined to form holoprotein. Ogawa and Shulman (1972) used this construct to demonstrate a direct connection between affinity state and heme structure by showing that the chemical shifts of the heme N M R peaks are altered by the addition of inositol hexaphosphate (IHP), an effector molecule known to stabilize the T state. This work and that of Cassoly and Gibson (1972) gave rise to the view
0 1994 American Chemical Society
Biochemistry, Vol. 33, No. 44, 1994
RR Spectroscopy of CNmethemoglobin Hybrids that the symmetrical valency hybrids are in the R state but can be switched to T by IHP addition. Thisview was reinforced by the recent work of Ackers and co-workers (1992), who have been able to measure the dimer-tetramer assembly free energies for all the ligation states accessible to the cyanomet hybrid system, using kinetic methods. For the symmetrical dicyanomet hybrids, they found the assembly free energy to be almost the same as for tetracyanometHb, -8.2 and -8.5 kcal/mol, respectively, consistent with the view that the symmetrical hybrids are predominantly R. They discovered, however, that the unsymmetrical hybrids, in which two cyanomet hemes are located in one ab dimer, have a much larger assembly free energy,-1 1.4 kcal/mol, halfway between the values for the symmetrical hybrids and deoxyHb (-14.4 kcal/mol). Thus, the free energy surface for ligand binding must depend upon the order in which the chains are filled. Ackers et al. (1992) suggested that theunsymmetrical diligated species remains in the T quaternary state. In this study, we provide new information on the tertiary and quaternary structures of the symmetrical cyanomet valency hybrids from resonance Raman spectroscopy. Laser excitation at 230 nm has been employed to enhance the vibrational bands of tyrosine and tryptophan residues, which have been shown (Rodgers et al., 1992) to monitor tertiary and quaternary interactions. In addition, the dependence on quaternary state of the important Fe-histidine stretching frequency has been reexamined with excitation in the visible region. The quaternary equilibrium is found to be nearly evenly balanced between the R and T structures, consistent with a diligand switch point when the ligands are bound symmetrically. On the other hand, IHP is found to stabilize the T state to only a modest extent for the valency hybrids, implying comparable affinity for both quaternary structures. In addition, IHP binding is found to influence the Fe-histidine stretching frequency of a subunits in both T and R states. In all cases, R state molecules containing deoxyhemes display a perturbed UVRR spectrum which resembles that of the recently discovered early transient of photodeligation (Rodgers & Spiro, 1994). This perturbation is believed to reflect motions of the helices lining the heme pocket, which lead to the quaternary rearrangement.
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spectroscopy was approximately 0.2 1 mM for (aFclllCNpFellC0)2 and 0.15 mM for (aFe11CO@Fe111CN)2 per tetramer. At these concentrations, the fraction of dimeric H b is less than 5% based upon the dimer-tetramer association rate constants determined by Smith and Ackers (1985). Spectroscopic measurements were done in a 0.01 M potassium phosphate, pH 7.0 buffer containing 0.5 mM NazEDTA and 10pM KCN. In experiments performed with IHP, the effector was present in a 10-fold molar excess with respect to the Hb tetramers. UVRR spectroscopy was carried out as previously described (Rodgers et al., 1992). Sample volumes were approximately 0.5 mL and were contained in a suprasil quartz NMR tube. The sample was rotated about a stationary helical coil to ensure complete mixing of the sample during spectroscopy. The temperature was kept between 7-10 "C by flowing cooled N2 gas across the sample tube. Samples were maintained as fullyligated or half-ligated forms by flowing CO or N2 gas, bubbled through H20, across the sample surface. Spectra result from 4 h of data collection. Each sample was in the laser beam for no longer than 2.5 h. Absorption spectra were measured before and after UVRR spectroscopy to check sample integrity. The quality of each UVRR spectrum, acquired over the period of an hour, was assessed by subtracting it from the first one-hour spectrum obtained with a fresh sample. If any discrepancies were observed the spectrum was discarded. Spectra were corrected for the response of the monochromator and the detector using a deuterium lamp. Difference spectra were generated using the internal intensity standard sodium perchlorate, which was present at a concentration of 0.2 M. Visible RR spectroscopy was performed using the 441.6nm line from a HeCd laser. Spectra were collected using a scanning double monochromator and detected with a photomultiplier tube. Sample conditions were the same as those used for UVRR spectroscopy. Curve fitting of the data was accomplished using 50% Lorentzian and 50% Gaussian line shapes with fixed widths of 15-17 cm-I. The adequacy of the fit was checked by examining x2values resulting from the deviations between the simulations and the actual data.
RESULTS
Fully-Ligated Hybrids Have R State UVRR Spectra. Figure 1 compares UVRR spectra for the CO adducts of the valency hybrids and of native hemoglobin, HbA. In all three HbA was prepared from fresh human blood by standard Hb species each heme bears a ligand, and the quaternary procedures (Antonini & Brunori, 1971). Theaandpsubunits state is therefore expected to be R. Excitation at 230 nm were separated following the procedure of Bucci and Fronticelli enhances Raman modes associated with tyrosine and tryp(1965) as modified by Yip et al. (1977). The purity of the tophan ring vibrations. These are labeled Y and W, subunits was determined by nondenaturing gel electrophoresis respectively, in Figure 1, with the mode numbers as assigned (Riggs, 1981). Full regeneration of the sulfhydryl groups previously (Harada & Takeuchi, 1986; Rava & Spiro, 1985). was assessed using the reagent N-ethylmaleimide. Isolated Many of these bands show altered intensities and/or frequena and p subunits were exposed to potassium ferricyanide and cies in deoxyHb; the deoxyHb minus HbCO difference potassium cyanide to generate the cyanomet derivative. The spectrum is shown at the bottom of Figure 2. This difference cyanomet chains were then passed through a Sephadex G-25 spectrum is specifically associated with R T quaternary column to remove potassium ferrocyanide and excess potassium structure changes (Rodgers et al., 1992). Arguments have cyanide. The presence of cyanide ligand bound to the heme been given for assigning the difference features mainly to the was checked using both absorption and visible resonance Tyr a42 and Trp 1837 residues, which are at the alp2 interface, Raman spectroscopy. The cyanomet chains were combined and which alter their hydrogen-bond status in the R-T with complementary chains containing ferrous heme CO transition adducts to create the symmetrical hybrids ( ~ F ~ I I I C N ~ ~ ~ I I C O ) ~ (Baldwin & Chothia, 1979). When UVRR spectra of the hybrid CO adducts are and (aFer1COpFe1I1CN)2.The hybrid tetramers were isolated subtracted from the HbCO spectrum, however, there are no using a CM-52 (Whatman) ion exchange chromatography spectral features above the noise level. Only the hybrid with column as previously described (Yip et al., 1977). All manicyanomet hemes in the @ chains, (aCOP+CN)2 is shown in pulations were performed at 4 OC. Photolysis of these CO Figure 1, but the same result was obtained with (a+CNPCO)*. adducts in an N2 atmosphere resulted in the formation of the This control experiment confirms that hybrid CO adducts are half-ligated forms. The concentration of the hybrids for
MATERIALS AND METHODS
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13134 Biochemistry, Vol. 33, No. 44, 1994
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FIGURE 1 : Comparison of UVRR spectra for fully ligated tetramers. The 230-nm excited RRspectrum of (A) HbCO, (B) (aFcrrrCNflFc11)2, (C) (aFc111CNflFc11)2 with 2.1 mM IHP, and (D) (aFc111CNflFc11C0)2 minus HbCO. The difference spectrum is multiplied by a y-scale with 2.1 mM IHP minus (aFC1l1factor of 2. (E) (&111CN@Fc11C0)2 CNflFe11C0)2.The difference spectrum is multiplied by a y-scale At factor of 2. Similar data were obtained for (aFC11C0flFe111CN)2. the Hbconcentrationsused, 0.15-0.25mM, thedegreeof dissociation into dimers is
