Biochemisrry 1991. 30. 1237-1 247 Nagai, K . ( I 9x9) Nature 336, 265-266. Ozaki, Y., Iriyama, K., Ogoshi, H., & Kitagawa, T. (1987) J . A m . Chem. SOC.109, 5583-5586. Perutz, M. F., Kilmartin, J. V., Nagai, K., Szabo, A,, & Simon, S. R. (1976) Biochemistry 15, 378-387. Privalov, P. L., Griko, Yu. V., Venyaminov, S. Yu., & Kutyshcnko, V . P. (1986) J . Mol. Biol. 190, 487-498. Puctt, D. (1973) J . Biol. Chem. 248, 4623-4634. Reid, L. S., Lim, A. R., & Mauk, A . G . (1986) J . Am. Chem. SOC.108, 8 197-8201, Ringe, D., Pctsko, G . A., Kerr, D. E.. & Ortiz de Montellano, P. R. (1984) Biochemistry 23, 2-4. Rougee, M., & Brault, D. (1973) Biochem. Biophys. Res. Coninrun. 55, 1364. Rougcc, M., & Brault, D. ( 1975) Biochemistry 14,4100-4 106. Rousscau, D. L., Ching, Y.-C., Brunori, M., & Giacometti, G. M . (1989) J . B i d . Chem. 264, 7878-7881. Sage, J . T., Morikis, D., & Champion, P. M . (1989) J . Chem. Phys. 90, 301 5-3032. Sage, J . T., Li, P., & Champion, P. M. (1991) Biochemistry (following paper in this issue). Sassaroli, M., & Rousseau, D. L. (1987) Biochemistry 26, 3092-3098. Schoniackcr, K. T.. Delaney, J. K., & Champion, P. M. ( 1 986) J . Chetlr. PhJ'.y. 85, 4240-4247. Shelnutt, J . A., Alston, K., Ho, J.-Y., Yu, N.-T., Yamamoto, T., & Rifkind, J . M. (1986) Biochemistry 25, 620-627.
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Shen, L, L.,& Hermans, J., Jr. (1972) Biorhemisfry 11, 1836-1 849. Shimada, H., & Caughey, W. S. (1982) J . Biol. Chem. 257, 11893-11900. Sibbett, S . S . , Loehr, T. M., & Hurst, J. K. (1986) Inorg. Chem. 25. 307-313. Spiro, T. G., & Burke, J. M. (1976) J . A m . Chem. Soc. 98, 5482-5489. Srajer, V., Schomacker, K. T., & Champion, P. M. (1986) Phys. Rec. Lett. 57, 1267. Srajer, V., Reinisch, L., & Champion, P. M. (1988) J . Am. Chem. Soc. I 10, 6656-6670. Tanford, C. (1970) Adc. Protein Chem. 24, I . Teraoka, J., & Kitagawa, T. (1 980) J . Phys. Chem. 84, 1928. Teraoka, J., & Kitagawa, T. (1981) J. Biol. Chem. 256, 3969. Traylor, T. G., Deardurff, L. A., Coletta, M., Ascenzi, P., Antonini, E., & Brunori, M. (1983) J . Biol. Chem. 258, 12147-1 21 48. Traylor, T. G., Koga, N., & Deardurff, L. A. (1985) J . Am. Chem. Soc. 105, 6504-6510. Wang, C.-M., & Brinigar, W. S. (1979) Biochemistry 18, 4960-4977. Warshel, A., & Weiss, R. M. (1981) J . Am. Chem. Soc. 103, 446-45 I . Yu, N.-T., & Kerr, E. A. (1988) in Applications of Raman Spectroscopy to Biological Systems (Spiro, T. G., Ed.) pp 39-95, Wiley, New York.
Spectroscopic Studies of Myoglobin at Low pH: Heme Ligation Kinetics? J. Timothy Sage, Pusheng Li, and Paul M. Champion* Department of Physics, Northeastern University, Boston, Massachusetts 021 15 Received May 14, 1990; Revised Manuscript Receiced October I I , I990 On the basis of the characterization of heme structure and ligation in equilibrium, we explore both proximal and distal ligation kinetics of myoglobin below p H 4. Upon photolysis of MbCO, a significant five-coordinate heme population is observed, with a n intact iron-histidine bond that persists on the time scale of CO rebinding. Incomplete CO photolysis is attributed to a rapidly exchanging minority population of four-coordinate hemes, which leads to fast (> 1 O l o s-') geminate recombination. T h e possible relevance of such a mechanism a t p H 7 is also noted. Using a novel experimental protocol, we observe the resonance Ranian spectrum of partially photolyzed M b C O a s a function of continuous wave illumination time ( 7 ) . Undcr cxtcnded illumination ( 7 35 ms a t p H 3.4), there is a loss of intensity in the v4 region of the Raman spectrum and the iron-histidine mode is bleached from the spectrum of the five-coordinate photoproduct. In the Fe-CO stretching region of the CO-bound fraction, the intensity of the 526-cm-' mode increases with 7 a t the expense of the 491-cm-' mode. These changes a r e interpreted as being due to replacement of the proximal histidine ligand under continuous illumination. Complete relaxation to the pure four-coordinate deoxy heme structure observed in equilibrium is not observed even a s 7 m , presumably since CO rebinding leads to acidification of the iron and its complexation with histidine. W e propose a kinetic model to account for our results and discuss the implications for previous low-pH kinetics measurements. ABSTRACT:
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M y o g l o b i n has been widely utilized as a model system for studying thc relationship between protein structure, dynamics, and function. Of particular interest is the means by which the protein modulatcs the rate of binding of small ligands such as CO to the heme. Motion of the iron into the heme plane is believed to make a significant contribution to the activation barrier that must be overcome upon ligand binding. The 'This work was supported by grants from N S F (87-16382) and N I H ( A M-35090).
0006-2960/9 1 /0430-1237$02.50/0
magnitude of this proximal contribution has been successfully described by a simpl? harmonic approximation to the iron out-of-plane motion (Srajer et al., 1988). Distal contributions to ligand binding rates are also likely to be important, and interactions of the bound ligand with the distal histidine are believed to play a key role (Moffat et al., 1979; Doster et al., 1982; Nagai et al., 1987; Olson et al., 1988; Braunstein et al., 1988; Morikis et al., 1989). Previous work has shown that ligand binding kinetics in myoglobin can be modulated by varying pH, and increased
0 199 1 American Chemical Society
1238 Biochemistry, Vol. 30,No. 5, 1991 C O binding rates below neutral pH have been attributed variously to distal cffccts involving the interaction of the bound ligand with the E7 histidine (Doster et al., 1982) or to proximal effects involving the rupture of the bond between the heme iron and thc F8 histidine (Giacometti et al., 1977, 1981; Coletta ct al.. 1985: Traylor et al., 1983). The proximal effects are thought to arise from the presence of four-coordinate hemes in which the iron is in the heme plane and the binding activation barricr is significantly reduced. Such effects are suggestcd by model compound studies, where protonation of the histidine ligand at low pH leads to rupture of the iron-histidine bond and cnhanccd C O binding rates (Geibel et al., 1975; Cannon et al., 1976: White et al., 1979). It is clear that an accurate understanding of pH-dependent kinetic effects requires a careful study of acid-induced structural changes. In the preceding paper (Sage et al., 1991), we used static spcctroscopic measurements to characterize structural changes a t the heme site due to the global conformational rearrangement of the protein that occurs below pH 4. In addition, we determined that the partially unfolded myoglobin conformation that is dominant below pH 4 is clearly distinct from the “open pocket” conformation that has been observed in MbCO’ above pH 4 (Morikis et al., 1989). Both of these conformations are expected to differ kinetically from the “closed packet” native conformation that is predominantly populated at neutral pH. Previous work has explored the ligand binding kinetics of different folded conformations above pH 4 (Doster et al., 1982; Ansari et al., 1987). Here, we present a preliminary kinetic characterization of the partially unfolded state observed below pH 4. Since very fast rebinding kinetics are expected for four-coordinate hemes, proximal ligation is a key issue. Although the iron-histidine bond is clearly broken for deoxyMb in equilibrium (Sage et al., 1991). photolysis of MbCO above pH 2.4 produces a five-coordinate species with this bond intact. Furthermore, this species remains stable on the time scale of CO rebinding. Under extended illumination, the iron-histidine stretching mode disappears from the resonance Raman spectrum of the photoproduct, but the iron remains five-coordinate. Complete photolysis of MbCO is prevented due to rapid (subnanosecond) geminate CO recombination. In order to explain this fast rebinding phase, we propose the existence of a minority population of four-coordinate hemes in rapid exchange with the observed majority populations of five-coordinate photoproducts. We present a model that describes the complex ligation kinetics observed and point out some pitfalls that should be avoided in interpreting kinetic data in the pH < 4 region.
THEORETICAL BACKGROUND On the basis of the results reported in the preceding paper (Sage et al., 1991), we present a simple framework for interpreting myoglobin structure and heme ligation states at low pH. In Figure 1 we expand the standard three-state model used to dcscribc binding and photolysis of exogenous ligands in myoglobin (Murray et al., 1988). In this model, the labels A, B, and C stand for the various states of the ligand with respect to the protein:
whcrc A indicates C O bound to the heme, B indicates C O in the heme pocket, and C represents M b with C O in solution. I Abbreviations: Mb, sperm whale myoglobin; Fe-His, iron-histidine; cw. continuous wavc; R R . rcsonance Raman.
Sage et al. histidine bound
native conformation
histidine free
matrix C O in
u,
acid CO bound conformation FIGURE 1: Scheme used for describing myoglobin structure and dynamics as a function of pH. A, B, and C represent the protein with
CO bound to the heme, with CO in the heme pocket but not directly bound, and without CO in the heme pocket, respectively. N and U represent the global state of the protein in the native and acid conformations, respectively, and + and - indicate the presence or absence of the bond between the heme and the proximal histidine.
The various rate constants involve photolysis ( k L ,where the spontaneous off rate is neglected), binding from the pocket to the heme (kBA), as well as the entry (kin) and exit (kOut) rates of the ligand between the solution and the pocket. Note that the bimolecular aspect of the problem is carried by the CO concentration dependence of kin = kin[CO] and that the protein concentration is assumed to be much less than [CO] so that kin is constant. One should also keep in mind that this simple model ignores intermediate states that have negligible populations in room temperature solutions and that the state denoted B here should not necessarily be interpreted as the (contact pair) B state observed in low-temperature geminate rebinding studies. The geminate states B (contact pair) and C (separated pair) observed at room temperature by Jongeward et al. (1988a) with ligands other than C O have been collapsed into a single geminate state, B, in the present simplified analysis. It should be recognized that the geminate rebinding rate, kBA,observed (Henry et al., 1983) for MbCO at room temperature is quite likely the product of the binding yield from the contact pair and the rate of contact pair formation from the separated pair. Since the overall amplitude of the geminate process in MbCO is only 4%, the amplitude for the direct decay of the contact pair is much too small to be detected and the three-state model is sufficient to describe all kinetic observables in the MbCO system a t neutral pH (Henry et al., 1983). As the pH is lowered and the rebinding rate from the contact pair is sufficiently increased, the amplitude for the direct decay from the contact pair should be detectable as an “unphotolyzed” fraction, with rebinding rates comparable to the laser photolysis rate. In the low-pH regime, the effective three-state analysis is still useful, bearing in mind that the states A, B, and C may need to be reinterpreted in line with the analysis of Jongeward et al. (1 988a). The generalization of the three-state model in Figure 1 is based on the structural characterization of myoglobin at low pH (Sage et al., 1991), which indicates that a complete understanding of ligand binding kinetics must allow for loss of the proximal histidine (+ = histidine present, - = histidine absent) as well as a partial loss of tertiary structure (N = native structure, U = “unfolded” structure). As might be expected, these pH-induced changes can profoundly affect the various rate constants in eq 1. At this stage, we digress briefly in order to clarify the meaning of Figure 1 in the context of previous work performed in the intermediate and physiological pH regime (4.5-7.0).
Spectroscopic Studies of Myoglobin a t Low p H
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Within this intermediate pH regime, we find that it is sufficient to consider only the states NA+,NE+,and Nc+, since the other statcs in Figurc 1 arc not significantly populated. It is important to rcalizc that each of the states NA+, NE+,and Nc+ are thenisclvcs composed of numerous substates with different protein conformations (Ansari et al., 1985). These different protein substates affect the iron-porphyrin equilibrium displaccmcnt and the local conformation of the distal pocket. For example, the opcn and closed forms of MbCO referred to earlier (Morikis et al., 1989) are both associated with the NA+ state and have been identified with the A, and A , CO conformers observed by using the infrared (Ansari et al., 1987) and Raman (Morikis ct al., 1989) marker frequencies of the FeCO moiety. The open conformation probably lies on the pathway toward the unfolded structure; nevertheless, its true significancc at physiological pH may be related to the ability of ligands to gain access to the heme binding site. The pH dependence of the A, and A , state populations suggests that the systcni is primarily in an open conformation below pH 4.5. Thus, for thc purpose of the low-pH analysis presented here (Le., pH < 4.5). we denote NA+as a single “state”. In principle this is an ovcrsiniplification, since all states should be considered simultaneously. However, given the complexity of the problem, wc believe it is reasonable to separate the analysis into low-pH (> k , , as might be the case for the ligand cntry rates (k,,,), even the small population of open form at pH 7 can play a significant role in the binding process. At low temperature, the average in eq 2 cannot be performed since the time scale for interconversion between the states is very slow. This leads to genuine inhomogeneities in the kinctic rcsponsc. For the geminate rebinding reaction there exist at Icast two, separate, broad barrier height distributions describing the heme rebinding rates, kBA(Doster et al., 1982). Wc bclicvc that thc breadth of these broad, finely grained, distributions is controlled by the proximal effects discussed
Stoppedflow (kL = 0 , Nc(0) = 1, NA(0) = NB(0) = 0 , kin > kBA+due ,to the absence of proximal constraint (Traylor et al., 1983; Srajer et al., 1988). To conclude this section, we summarize the solutions to the simple kinetic scheme of eq 1 under various experimental conditions. We always consider the limiting case kin
