spin state on the heme orientational preference in myoglobin

Vo1.158, No. 2,1989 Janua~ 31,1989

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PROTON NMR STUDY OF THE INFLUENCE ON IRON OXIDATION/LIGATION/SPIN STATE ON THE HEME ORIENTATIONAL PREFERENCE IN MYOGLOBIN Gerd N. La Mar*, Wanda S. Smith, Nicolette L. Davis, David L. Budd, and Mark J. Levy Department of Chemistry, University of California Davis, California 95616 Received November

14, 1988

Proton nuclear magnetic resonance spectrosopy has been utilized to demonstrate that the degree of heme orientational disorder within a given myoglobin protein matrix can be a sensitive function of the oxidation/ligation/spin state of the heme iron. For sperm whale deuterohemin-reconstituted myoglobin, the equilibrium was found to strongly favor (5.7 to 7.8 kJ/mol) the X-ray characterized heme orientation in all six-coordinate states, but with a considerable reduction in preference (to 1.6 kJ/mol) in the five-coordinate deoxy state. In native yellow fin tuna myoglobin, changes in heme orientational preferences of ~3 kJ/mol occur even between two sixcoordinate ferric states differing solely in spin states. ~ 1989Ao~demicP..... ~no.

The detailed structures of the heme cavity of b-type hemoproteins initially were considered unique. More recent solution NMR studies, however, revealed that, not only are there often significant equilibrium populations of an alternate heine orientation rotated by 180 ° about the ~,y-meso axis from that characterized in single crystals (A, B in Figure 1), but that those alternate heme insertion isomers are formed equally probably in the initially reconstituted complex (1-5). Thus 1H NMR spectroscopy has found most of the common myoglobins, Mb, (1-3) and hemoglobins, Hb (4), as well as cytochrome b5 (5), with rotationally disordered hemes at equilibrium, with the population of the alternate form ranging from a few percent to essentially completely disordered systems (Chironomus Hb, 40% (6); rat cytochrome b5, 45% (unpublished data, Pochapsky, T., Sligar, S., La Mar, G.N., and Lee, K.-B.)). The functional consequences of such disorder varies, with sperm whale Mb exhibiting a negligible change in CO affinity (7,8), while Chironomus Hb * To whom correspondence should be addressed.

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0006-291X/89

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displays a large change in the Bohr effect (9), and cytochrome b5 a ~30 mv difference in redox potential (10). The heme orientational disorder is most readily detected, structurally characterized and quantified via 1H NMR spectroscopy of the low-spin ferric state (1-6). More recent work has shown that differences in the degree of disorder in freshly reconstituted and equilibrated Mb can also be detected by circular dichroism studies, and that the sign of the ellipticity at equilibrium, like the heme methyl contact shift pattern in metMbCN 1H NMR spectra, leads to the solution determination of the absolute heme orientation (11). It is currently assumed that heme orientationaI disorder is a property of a given genetic variant and depends on the specific contacts of the heme side chain groups at positions 2 and 4 within a folded polypeptide chain (3). We demonstrate herein for two myoglobin complexes that the degree of equilibrium disorder is not an intrinsic property of a protein but depends on the state of the heme iron. We focus on two systems for which the necessary heine resonance assignments have been reported on the basis of isotope labeling that clearly define the disorder in the cyano met complexes, deuteroheminreconstituted sperm whale Mb (1) and native yellow fin tuna Mb (3). EXPERIMENTAL Protein preparation. Sperm whale myoglobin was obtained as a lyophilized powder from Sigma and was used as received. Yellow fin tuna muscle was obtained from Starkist. Isolation of the protein was carried out as described previously (12). Samples for NMR were 1-3 mM in protein in pH 7 phosphate buffer prepared in 2 H 2 0 . Preparation of apo protein and reconstitution were carried out as described earlier (2), except that apo protein from yellow fin tuna myoglobin was not lyophilized. R e d u c t i o n of met-aquo myoglobin was carried out in a glove box by the addition of two equivalents of a freshly prepared, degassed solution of sodium dithionite (Nakarai Chemicals) to protein in 0.2 M NaC1/2H20 in a 5mm NMR tube which had been exhaustively degassed and thoroughly flushed with nitrogen. The pH of the sample was adjusted in the glove box. In order to remove excess dithionite and products of reduction without autoxidation, the sample was converted to the carbon monoxy form and eluted from a G-25 Sephadex column. Oxidation of deoxy myoglobin was carried out by the addition of two equivalents of potassium ferricyanide and excess potassium cyanide, followed by elution from a G-25 Sephadex column. C y a n i d e a b s t r a c t i o n was effected by the addition of five equivalents of a solution of hydroxocobalamine (Sigma), buffered at pH 7, to the protein in the same buffer, followed by incubation at room temperature for two hours. The excess hydroxocobalamine and reaction products were removed by chromatography with G-25 Sephadex. 463

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P r o t o n N M R s p e c t r a were recorded on Nicolet NT-200 (4096 data points over 20 KHz) and Nicolet NT-360 (8196 points, over 10 KHz) spectrometers operating at 200 or 360 MHz, respectively. The residual water peak was eliminated by a presaturation pulse. RESULTS The 1H NMR trace of sperm whale deuterohemin-metMbCN at equilibrium is shown in Figure 1C; the set of peaks Ai, Bi have been shown to arise from the orientations shown in A and B, respectively, in Figure 1, with an equilibrium A:B ratio ~16:1 (1). Upon reduction with dithionite, the 1H NMR trace shown in Figure 2D results, for which the

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FIGURE 1: (A) Heme orientation as found in the X-ray crystal structure; (B) as rotated by 180° about the ~,T meso axis. The plane of the proximal histidine F8 and the position of Phe CD1 are indicated. R = vinyl in native hemin, hydrogen in deuterohemin. (C-F) Low-field resolved portions of the 200 MHz 1H NMR spectra of sperm whale deuterohemin-Mb in 0.2M 2H20, 37°C, pH 6.3. (C) Equilibrated metcyano complex with assigned heine methyl peaks Ai, Bi corresponding to heme orientations in Figures 1A, 1B, respectively. (D) Deoxy Mb complex resulting from treating sample in trace C with dithionite; peaks ai, bi arise from pyrrole 2-H and 4-H (14). (E) Deoxy complex from trace D after reaching equilibrium, with ai:bi ~2:1. (F) Regeneration of the met-cyano complex by treating sample in trace E with ferricyanide and cyanide. 464

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C

D

II II II II

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l

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FIGURE 2: Low-field portions of the 360 MHz 1H NMR spectra of yellow fin tuna Mb, 25°C, pH 6.5 in 0.1M phosphate buffer in D20. Resolved heme methyl peaks are labelled Ai, Bi in the metMbCN spectra, and ai, bi in the metMbH20 spectra (3), corresponding to the orientations shown in Figures 1A and B, respectively. (A) Equilibrated metMbCN with Ai:Bi ratio -8:1 (3). (B) metMbH20 spectrum resulting after rapid stripping by hydroxocobalamine of cyanide from sample in trace A. (C) Sample of metMbH20 from trace B after reaching equilibrium; two methyl peaks B1, B2 are degenerate (3). (D) Regeneration of metMbCN complex by adding excess cyanide; note Ai:Bi ratio is now -2:1.

characteristic pyrrole 2-H, 4-H peaks a], a2 and bl, b2 reflect the ratio of components seen in the met-cyano state. With time, however, peaks bl and b2 increase in intensity at the expense of peaks a 1 and a2, reaching a final A:B ratio of -2:1, as shown in Figure 1E. Reoxidation of the deoxy sample in Figure 1E with ferricyanide in the presence of excess C N - generates the trace in Figure 1F, demonstrating the -2:1 relationship between the ai and bi peaks for the two heme orientations of the deoxy derivatives. Within 24 hours, the trace of Figure 1F has reverted to its equilibrium condition as found in Figure 1C. 465

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Heme RotationalEquilibrium Constants, 25oc in 2H2Oa Oxidation state Fe(III) Fe(III) Fe(llI) Fe(III)

Sixth Ligand H20 N3" CNOH-

Fe(n) Fe(U)

CO

Sperm whale deuterohemin-Mb pH Keq 6.2 19 + 4 7.0 16_44 8.0 19+4 10 16+ 4 12 1.7 + 0.2 7.0 1.9 + 0.2 7.2 14-44

Yellowfin tuna Mb pH Keq 6.5 2.0 + 0.5 8 8+2 11 2.0 + 0.5 9

8+2

a) Keq is the equilibrium intensity ratio of peaks Ai:Bi or ai:bi corresponding to orientations A and B in Figure 1.

B y equilibrating deuterohemin-Mb complexes in the presence of other ligands in either the ferric or ferrous state, then converting the samples quickly to the met-cyano state by addition of C N - a n d ferricyanide (for ferrous samples), it was possible to determine the equilibrium ratio of orientations for those complexes from the relative areas: of the A1 to B1 peaks in the metMbCN spectrum. The resulting data are included in Table I. The equilibrium ratio of heine orientations A:B in yellow fin tuna metMbCN has been reported as 8:1 (3), as shown by the ratio of assigned peaks Ai:Bi in Figure 2A. When cyanide is stripped rapidly from this sample by hydroxocobalamine, the resulting m e t M b H 2 0 trace initially establishes the same set of peaks ai:bi -8:1 (peaks b l , b2 are degenerate (3)). With time, peaks bi grow as peaks ai decrease in intensity until the trace in Figure 2C is obtained at equilbrium, with an ai:bi ratio - 2 : I . Upon displacing the bound water with excess cyanide, the trace in Figure 2D is obtained which confirms the 2:1 ratio of isomers in the met-aquo sample. Within a few days, the trace in Figure 2D reverts back to the one displayed in Figure 2A. Data on the equilibrium ratio of heme orientation for yellow fin tuna for other oxidation/ligation ~ states,, detected by integrating the cyano-met peak upon converting rapidly to metMbCN, are also included in Table i. DISCUSSION The data in Figures 1 and 2, as well as Table I , clearly demonstrate that the equilibrium between the two heme orientations depends on the state o f the heme iron. In sperm whale deuterohemin-Mb, the equilibrium constant strongly favors the native X-ray characterized 466

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orientation (13) by 5.7-7.8 kJ/mol in all five- and six-coordinated states, whether ferric or ferrous, high-spin or low-spin. At very alkaline pH (>11.5), sperm whale deuterohemin-metMbOH exhibits a strongly reduced preference (Table I), as previously reported for the native protein ( t l ) , but may result from a major pH induced transition on partial unfolding at this extreme condition. On the other hand, the unique five-coordinate sperm whale deoxy deuterohemin-Mb displays much less preference, ~1.6 kJ, for the X-ray characterized orientation. Yellow fin tuna provides a more discriminating environment for the heme among the ligated, six-coordinated states. Thus the two states, met-cyano and met-aquo, which exhibit the same preference in both deuterohemin-reconstituted and native sperm whale Mb (1,2), yield differences in preference of ~3 kJ/mole. We were unable to determine the equilibrium constant for the heme orientation in native yellow fin tuna deoxy Mb because of the much slower rate of heme reorientation for protohemin than deuterohemin and the much greater autoxidizability of yellow fin tuna deoxy Mb (12). We note that the present data reverse the earlier tentative conclusion that differences in relative populations of heme orientations in the yellow fin tuna metaquo Mb and equilibrated metMbCN may represent a functional role i_.~n vivo for the metastable state of the Mb (3). The two cases described here present contrasting sensitivity of the preference of the heme orientation to the oxidation/ligation/spin state of the heine iron. This indicates that it is not possible at this time to generalize about the equilibrium structural homogeneity in the heme pocket for other states of the protein based on detailed characterization of heme rotational disorder in any one oxidation/ligation/spin state. Moreover, this suggests that the degree of heme disorder is likely to be a function of metal substitution. It has been shown that the sensitivity to functional properties of the two heine orientations is similarly unpredictable, clearly manifesting itself in azide binding preference in Hb A (4) and yellow fin tuna Mb (La Mar, G.N., Smith, W.S., Levy, M.J., unpublished results), but not in sperm whale Mb, and manifesting itself in CO affinity for Chironomus Hb (9) but not in sperm whale Mb (7,8). The variable heine rotational preferences with oxidation/ligafion/ spin states and the variable functional sensitivity of the two heme orientations for different homologous proteins infer that there exists fine-tuned coupling between equatorial and axial protein-heme interactions. The detailed elucidation of the basis of this variable coupling must await the results of planned solution NMR studies of the structural consequence of the two alternate heme orientations. 467

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ACKNOWLEDGMENT This research has been supported by a grant from the National Institutes of Health, HL-16087.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

La Mar, G.N., Budd, D.L., Viscio, D.B., Smith, K.M., and Langry, K.C. (1978). Proc. Nat. Acad. Sci., USA 75, 5755-5759. La Mar, G.N., Davis, N.L., Parish, D.W., and Smith, K.M. (1983). J. Mol. Biol. 168, 887-896. Levy, M.J., La Mar, G.N., Jue, T., Smith, K.M., Pandey, R.K., Smith, W.S., Livingston, D.J., and Brown, W.D. (1985). J. Biol. Chem. 260, 13694-13698. Yamamoto, Y., and La Mar, G.N. (1986). Biochemistry. 25, 5288-5297. La Mar, G.N., Bums, P.D. Jackson, J.T., Smith, K.M., Langry, K.C., and Strittmatter, P. (1981). J. Biol. Chem. 256, 6075-6079. La Mar, G.N., Smith, K.M., Gersonde, K., Sick, H., Overkamp, M., (1980). J. Biol. Chem. 255, 66-70. Light, W.R., Rohlfs, R.J., Palmer, G., Olson, J.S. (1987). J. Biol. Chem. 262, 46-52. Aojula, H.S., Wilson, M.T., and Morrison, I.E.G. (1987). Biochem. J. 243, 205-210. Gersonde, K., Sick, H., Overkamp, M., Smith, K.M., and Parish, D.W., (1986). Eur. J. Biochem. 157, 393-404. Walker, F.A., Emrick, D., Rivera, J.E., Hanquet, B.J., and Buttlaire, D.H., (1988). J. Am. Chem. Soc. 110, 6234-6240. Aojula, H.S., Wilson, M.T., Moore, G.R., and Williamson, D.J. (1988). Biochem. J. 250, 853-858. Livingston, D.J., Watts, D.A., and Brown, W.D. (1986). Arch. Biochem. Biophys. 249, 106-115. Takano, T. (1977). J. Mol. Biol. 110, 537-568. Jue, T. and La Mar, G.N. (1984). Biochem. Biophys. Res. Comm. 119, 640-645.

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