VOL. 18, NO. 24, 1979
CO AND N O BINDING TO MYOGLOBIN AND MODEL
Messana, C., Cerdonio, M., Shenkin, P., Noble, R. W., Fermi, G., Perutz, R. N., & Perutz, M. F. (1978) Biochemistry 17, 3652. Morimoto, H., & Kotani, M. (1966) Biochim. Biophys. Acta 126, 176. Ogoshi, H., Watenabe, E., Yoshida, Z., Kincaid, J., & Nakamoto, K. (1973) J. A m . Chem. SOC.95, 2845. Olafson, B. D., & Goddard, W. A. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1315. Ozaki, Y., Kitagawa, T., & Kyogoku, Y. (1976) FEBS Lett. 62, 369. Perutz, M. F., Heidner, E. J., Ladner, J. E., Beetlestone, J. G., Ho, C., & Slade, E. F. (1974) Biochemistry 13, 2187. Perutz, M. F., Sanders, J. K. M., Chenery, D. H., Noble, R. W., Pennelly, R. R., Fung, L. W.-M., Ho, C., Giannini, I., Porschke, D., & Winkler, H. (1978) Biochemistry 17, 3640. Smith, D. W., & Williams, R. J. P. (1968) Biochem. J. 110, 297. Smith, D. W., & Williams, R. J. P. (1970) Struct. Bonding (Berlin) 7, 1 .
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Spaulding, L. D., Chang, C. C., Yu, N.-T., & Felton, R. H. (1975) J. A m . Chem. SOC.97, 2517. Spiro, T. G. (1975) Biochim. Biophys. Acta 416, 169. Spiro, T. G., & Burke, J. M. (1976) J. A m . Chem. SOC.98, 5482. Spiro, T. G., Stong, J. D., & Stein, P. (1979) J. Am. Chem. SOC.101, 2648. Strekas, T. C., & Spiro, T. G. (1974) Biochim. Biophys. Acta 351, 237. Strekas, T. C., Adams, D. H., Packer, A. J., & Spiro, T. G. (1974) Appl. Spectrosc. 28, 234. Takano, T. (1977a) J. Mol. Biol. 110, 537. Takano, T. (1977b) J. Mol. Biol. 110, 569. Warshel, A. (1977a) Annu. Rev. Biophys. Bioeng. 6, 273. Warshel, A. (1977b) Proc. Natl. Acad. Sci. U.S.A.74, 1789. Williams, R. C., Jr., & Tsay, K.-Y. (1973) Anal. Biochem. 54, 137. Yonetani, T., Iizuka, T., & Waterman, M. R. (1971) J . Biol. Chem. 246, 7683. Yu, N.-T. (1977) CRC Crit. Rev. Biochem. 4, 229.
Nitric Oxide and Carbon Monoxide Equilibria of Horse Myoglobin and (N-Methylimidazo1e)protoheme. Evidence for Steric Interaction with the Distal Residuest Robert W. Romberg and Richard J. Kassner*
ABSTRACT: The Soret absorption maxima and extinction coefficients of the CO and N O complexes of horse myoglobin and (NMe1m)protoheme (NMeIm = I-methylimidazole) have been determined. The partition coefficient N, equal to the ratio PlI2(CO)/PII2(NO),has been determined spectrophotometrically for horse myoglobin and (NMe1m)protoheme. (NO) values calculated from the partition coefficients are 5.7 X lo-' mmHg for (NMe1m)protoheme and 1.1 X lod mmHg
for horse myoglobin. The ratio of Pl12(NO)values for protein and model is 1.9 which is similar to a value of 1.6 reported for the ratio of P1/2(02)values. These values may be compared to a ratio of 15 for C O binding to protein and model complexes. This different ratio for CO provides further evidence for steric interaction of the bound CO with the protein based on a consideration of the preferred nonlinear geometry of Fe-NO and Fe-02 and the linear geometry of Fe-CO.
D a t a on infrared stretching frequencies have been extremely important in revealing the nature of protein ligand bonds. Over the last several years the literature has provided CO infrared stretching frequencies of various hemoproteins (Caughey, 1970; Caughey et al., 1969; Alben & Caughey, 1968). Many normal hemoglobins exhibit similar CO frequencies, but abnormal hemoglobins with the distal histidine replaced by amino acid residues bearing hydrophilic side chains have considerably higher CO frequencies (Caughey et al., 1969). The stretching frequencies of these mutant hemoglobins approach the uco of model heme complexes (Collman et al., 1976). Differences in the uco have been interpreted in terms of the structure and ligand binding properties of hemoprotein and model complexes. The Fe-CO bond has been found to be linear in the nonhindered model systems Fe(TpivPP)(NMe1m)CO' (Hoard, 1975) and Fe(TPP)(py)CO (Peng & Ibers, 1976). X-ray data (Huber et al., 1970; Padlan & Love,
1974) indicate that in proteins the CO to Fe bond is bent or tilted. A neutron diffraction study has indicated a Fe-C-0 bond angle of 135' in horse myoglobin (Norvell et al., 1975). These studies indicate that amino acid residues in the heme pocket are close enough to cause CO to bend or tilt from its preferred linear structure. Specifically, valine (residue E 1 1) and the distal histidine (residue E7) are capable of interacting with CO when it is bound to horse myoglobin (Norvell et al., 1975). Caughey has attributed the lower urn in normal hemoglobins and myoglobins to a donation of electron density from the nitrogen of the distal histidine to the partially electropositive carbon atom of the bent or tilted CO molecule (Caughey, 1970). Collman has suggested that the lower stretching frequencies result from steric hindrance which distorts the C O from its preferred linear structure (Collman et al., 1976).
'From the Department of Chemistry, University of Illinois at Chicago Circle, Chicago, Illinois 60680. Receiued May 7, 1979; reuised manuscript received Seplember 12, 1979.
0006-2960/79/0418-5387$01 .OO/O
'
Abbreviations used: NMeIm, 1-methylimidazole; PP, protoporphyrinate; TpivPP, meso-tetrakis(a,a,a,a-o-pivalamidopheny1)porphyrinate; NTrIm, 1 -tritylimidazole; py, pyridine; partial pressure of gas at half-saturation; Mb, myoglobin; Im, imidazole; TPP, meso-tetraphenylporphyrinate;CTAB, cetyltrimethylammonium bromide.
0 1979 American Chemical Society
5388
BIOCHEMISTRY
R O M B E R G AEiD K A S S N E R
In contrast, theoretical calculations using FePP and NMeIm (NMeIm)CO, oxygen was rigorously excluded. C O was show that oxygen should be bound in a bent geometry (Kirpassed over the aqueous solution of Fe111PP(NMeIm)2for 2 chner & Loew, 1977). X-ray structural analysis has shown h and subsequently bubbled through the solution for 30 min this is to be the case in a nonrestricted model heme complex prior to the addition of 0.0005 g of ascorbate from the side (Fe-0-0 angle 135') (Collman et al., 1974; Jameson et arm. C O was then bubbled through the solution for 1 addial., 1978). It is well-known that bound 0, in the hemoproteins tional h, yielding Fe"PP(NMe1m)CO. is bent (Rodley & Robinson, 1972; Barlow et al., 1973; For the preparation of Fe"PP(NMeIm)NO, NO was bubPhillips, 1978; Makinen et al., 1978). It has therefore been bled through a solution of Fe"PP(NMe1m)CO until the comproposed that steric interactions decrease the CO affinity of plex was completely converted to the nitrosyl form as evidenced hemoproteins, while not altering the O2 affinity of hemoby UV-visible spectroscopy. We were unable to prepare proteins (Caughey, 1970; Antonini & Brunori, 1971, pp 91-93; Fe"'PP(NMe1m)NO; N O reduced Fe111PP(NMeIm)2 to Heidner et al., 1976; Peng & Ibers, 1976; Collman et al., Fe"PP(NMe1m)NO over a period of several hours without 1976). X-ray data indicate that the Fe-NO bond is also bent the apparent intermediate formation of the ferric nitric oxide complex. in model systems (Perry, 1968; Piciulo et al., 1974). Infrared frequencies (Maxwell & Caughey, 1976) and EPR spectra Preparation of Carbonyl- and Nitrosylmyoglobin. For the (Barlow & Erecinski, 1979) of hemoproteins are consistent preparation of carbonylmyoglobin, C O was passed over an with a nonlinear Fe-NO bond. If nitric oxide is bent with a aqueous solution of myoglobin containing 0.4 M phosphate bond angle similar to 0 2it, should not be affected by steric buffer, pH 7.0, at 22 "C in the thunberg cuvette for 12 h after hindrance in hemoproteins. C O , on the other hand, would which 0.0005 g of ascorbate was added from the side arm. CO prefer to bind in a linear geometry and would thus appear to was then passed over the solution for an additional 12 h until experience significant steric hindrance in binding to the hemopure carbonylmyoglobin was obtained. For the preparation proteins. It is our hypothesis that the ratio of the O2 affinity of ferrous nitrosylmyoglobin, N O was bubbled through the of Mb to the O2affinity of a model, P1/2(02)~~/P1/2(02)m~e~r solution of the carbonylmyoglobin until the carbonyl complex should be similar to the ratio of the N O affinity of Mb to the was completely converted to the nitrosyl form. NO affinity of a model, Pli2(NO)Mb/PI12(NO),~~,. A comPreparation of Nitric Oxide Metmyoglobin. For the prepparison of the affinities of the hemoproteins and the models, aration of NO metmyoglobin, a solution of horse heart metin terms of Pli2values, for CO, 02,and N O may indicate the myoglobin in 0.4 M phosphate buffer, at pH 7.0, 22 OC, was deaerated with nitrogen and then bubbled rapidly with N O extent to which steric hindrance affects CO binding to proteins. for 3 min to ensure complete complexation, and the spectrum The present work describes the measurement of the N O afwas recorded immediately thereafter. The resulting solution finities of a (1 -methylimidazole)protoheme complex and of gave a visible absorption spectrum that corresponded to the horse myoglobin. NO metmyoglobin complex reported by Wittenberg et al. Materials and Methods (1967). Upon standing for 5 h, the solution showed spectral Horse heart myoglobin (Type 111), crystalline bovine hemin changes characteristic of the conversion of myoglobin from (FePP) (Type I), and 1 -methylimidazole (NMeIm) were the Fe(II1) form to the Fe(I1) form. It has been reported that purchased from Sigma Chemical Co. Ultra pure carbon N O slowly reduces metmyoglobin (Drabkin & Austin, 1935). monoxide, chemically pure nitric oxide, and a 403-ppm nitric The same N O myoglobin spectrum was obtained by adding oxide in nitrogen gas mixture were obtained from Matheson. NO to carbonylmyoglobin. All other chemicals used were of reagent grade. A Matheson Determination of NO Affinities. CO and 403-ppm NO in Model 7372T gas proportioner with Kel-F packing equipped N 2 were mixed via the calibrated gas proportioner and the with No. 600 flowmeter tubes was used to prepare mixtures individual flows were adjusted appropriately to obtain the of CO and NO. The gas proportioner has a variable flow rate desired ratios of P ( C 0 ) and P(N0). The gas mixture was ranging from 4 to 57 cm3/min for both carbon monoxide and bubbled through a solvent saturation tower containing either nitric oxide in nitrogen as calibrated in our laboratory. water (for myoglobin solutions) or 20% NMeIm in water, to Thunberg tubes with 19/22 ground glass fittings from Lab eliminate sample concentration changes due to solvent evapoGlass were converted to thunberg cuvettes by replacing the ration upon bubbling. The solvent-saturated gas mixture was lower half of the tube with square I-cm i.d. glass tubing. A then bubbled into the solutions containing the heme carbonyls glass socket for a rubber septum was added on the top of the by using a 20-gauge stainless steel needle. Absorbance readside arm to permit the introduction of gases. Absorption ings were taken after raising the needle above the solution. spectra were recorded on a Cary 14R spectrophotometer. Absorbances were monitored at 418 nm for the model heme Measurements of pH were made with a Radiometer PHM 64 complexes and at 424 nm for the myoglobin complexes. Sixty pH meter. minutes of bubbling was generally required to obtain a constant Preparation of FePP(NMeIm)2Complexes. Hemin was absorbance. If after an additional 30 min of bubbling the dissolved in an aqueous solution containing 20% v/v NMeIm absorbance had not changed, the system was considered to be and the pH was adjusted to 9.0, yielding Fe111PP(NMeIm)2. at equilibrium. Bubbling occasionally resulted in the denatuThis concentration of NMeIm was found necessary to give only ration of some of the myoglobin. This could be prevented by the six-coordinate Fe"PP(NMe1m)NO complex in the preslowering the flow rate and limiting the bubbling time. Only ence of NO. At lower concentrations of NMeIm, there is experiments which exhibited good isosbestic points were used evidence for the formation of the five-coordinate Fe"PPN0 for calculations. complex in the presence of N O in keeping with previous obResults servations (Maxwell & Caughey, 1976). Deaeration of a Absorption spectra were first measured for all possible solution of FelllPP(NMeIm)zwith nitrogen for 1 hour, followed by addition of dithionite from the side arm, yielded species of model and hemoprotein complexes which are associated with the ligand binding equilibria in solution. Figure Fe11PP(NMeIm)2. 1A shows the Soret region of the absorption spectra of Preparation of Fe"PP(NMeIm)CO and Fe"PP(NMeIm)Fell*PP(NMeIm)z,Fe11PP(NMeIm)2,Fe"PP(NMeIm)CO, NO. In the preparation of the six-coordinate Fe"PP-
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CO A N D N O B I N D I N G TO M Y O G L O B I N A N D MODEL
Table I: UV-Visible Absorption Maxima and Extinction Coefficients for (NMe1m)protoheme and Myoglobin Complexes (NMe1m)protoheme hmax (nm)
(mM" cm-')a
41 1.5' 424.0' 419.0 415.0
156 210 219 126
E
form Fe(II1) Fe(I1) Fe"C0 Fe"N0 Fe"'N0
myoglobin E
(nm)
A,,
409.5 435.0 423.5 421.5 420.5
none
(mM-'
cm-')b
186 121 207 147 155
Extinction coefficients were based on the concentration of heme determined by the pyridine hemochrome method described Extinction by Falk (1964) usingan E = 191.5 mM-' cm-'. coefficients were calculated by using an E = 121 mM-' cm-' for deoxymyoglobin (Antonini & Brunori, 1971, p 19). 'Fe(PP)(NMeIm),. a
Table 11: Binding of NO to (NMe1m)protoheme at Different Partial Pressures of CO and NOa
~~
400
410
420
430
440
450
W A V E L E N G T H (nml
1
0
4050 3810 2250 1330 750 225
33.3 35.9 48.7 60.9 73.4 90.0
2030 2130 21 30 2070 2060 2030
a At 22 "C in 20% v/v NMeIm at pH 9.0. The partial pressure of CO divided by the partial pressure of NO. N is defined as
'
~FerlPP(NMeIm)NOIP(CO)/[ [Fe"PP(NMeIm)CO]P(NO) 1.
07
06 Lu
0
400
410
420
430
440
450
W A V E L E N G T H inrn)
1: (A) Soret absorption spectra of protoheme complexes in 20% NMeIm, p H 9.0,0.40 M hosphate buffer, and 22 OC. [Protoheme] = 3.6 X 10" M. (a) Fe' RPP(NMeIm),; (b) Fe"PP(NMeIm),; (c) Fe"PP(NMe1m)CO; (d) Fe"PP(NMe1m)NO. (B) Soret absorption spectra for horse myoglobin, pH 7.0,0.40 M phosphate buffer, and 22 OC. [Myoglobin] = 4.1 X 10" M. (a) Metmyoglobin; (b) myoglobin; (c) C O myoglobin; (d) N O myoglobin; (e) N O metmyoglobin. FIGURE
and Fe"PP(NMe1m)NO. Corresponding absorption maxima are recorded in Table I. Figure 1B shows the Soret region of.the absorption spectra of Fe"'Mb, FeIIMb, Fe"MbC0, Fe"MbN0, and Fe"'MbN0. By use of an extinction coefficient of 121 mM-' cm-' for oxymyoglobin, the extinction coefficients were calculated for Fe"'N0 myoglobin and Fe"N0 myoglobin. The corresponding absorption maxima and extinction coefficients are recorded in Table I for comparison to those of the model heme complexes. Absorption maxima and relative extinction coefficients for FeI'Mb, Fe"'Mb and carbonylmyoglobin are in good agreement with those previously reported (Antonini & Brunori, 1971, p 19). The differences between the ferrous carbonyl and nitrosyl compounds were much greater in the Soret region than in the visible region; for this reason the Soret region was chosen to follow absorption changes in the equilibration of the carbonyl
:
04
d
3
g
