Study Ofan Oxygenated Heme Complex in Frozen Solution

Proc. Nat. Acad. Sci. USA Vol. 69, No. 9, pp. 2396-2399, September 1972

Study of an Oxygenated Heme Complex in Frozen Solution by Mossbauer Emission Spectroscopy (oxygen binding/hemoglobin/Co(II)-protoporphyrin IX) LINDA MARCHANT*, MICHAEL SHARROCKt, BRIAN M. HOFFMAN*, AND ECKARD MtfNCKt * Department of Chemistry, Northwestern University, Evanston, Illinois 60201; and t Department of Physics, University of Illinois, Urbana, Ill. 61801 Communicated by I. C. Gunsalus, May 31, 1972

ABSTRACT We have used Mossbauer emission spectroscopy to study an oxygenated heme complex, produced in a N N frozen solution by nuclear decay from the isomorphous 57Co-labeled compound. The Missbauer parameters agree with those obtained by Mossbauer absorption spectroscopy of oxyhemoglobin. Thus, a nonprotein environment Co- 57Fe for iron can duplicate the unique oxyhemoglobin Moss- IV bauer spectrum. The electronic structure of the heme iron \ HEME,// 0 in oxyhemoglobin is not significantly influenced by the rn PLANE 02 protein environment. Miissbauer emission spectroscopy can be useful in the investigation of heme proteins. FIG. 1. Formation of 57Fe-labeled oxyheme-(OMe)2(C6H5N) by nuclear decay of 57Co in oxyCoP-(OMe)2(C5HsN). One aim of the study of hemoproteins is to learn the mecha- nisms by which an apoprotein can modify the chemical re- activity and the electronic structure of the heme prosthetic MOSSBAUER EMISSION SPECTROSCOPY group. Recent studies of oxygen binding to cobalt proto- Observation of the Mbssbauer effect can provide information porphyrin, both in solution and incorporated into an apopro- about the chemical environment of the 57Fe nucleus by either tein, have achieved a partial understanding of the influences emission or absorption spectroscopy. Both techniques re- of a protein on oxygen binding. Like the heme group in hemo- quire the same basic experimental arrangement: a source globin and myoglobin, five-coordinate complexes of the heme containing 57Co, an absorber containing 57Fe, and a means analog Co(II)-protoporphyrin IX dimethyl ester [CoP- of counting radiation transmitted through the absorber. (OMe)2], in solution can bind molecular oxygen (1, 2). When As shown in Fig. 2a, 57Co decays by electron capture to 57Fe, Co(II)-protoporphyrinIX (CoP) is incorporated into apohemo- populating an excited state (T11, = 0.1 Msec) of the iron globin or apomyoglobin, it has a greater affinity for oxygen nucleus. The 14.4-keV 7y-radiation emitted in the subsequent and a greater resistance to oxidation than free [CoP-(OMe)2]T de-excitation process can resonantly excite 5"Fe nuclei in (1, 3, 4). The electronic structures, however, of CoP and the absorber. In the absorption technique, the source emits its oxygen adduct in a protein environment do not differ a monochromatic y-radiation, and the hyperfine energies significantly from those of CoP-(OMe)2 and its oxygen adduct of the nuclear levels of 57Fe in the absorber are measured in free solution, as evidenced by optical absorption and elec- (Fig. 2b). Emission spectroscopy, on the other hand, uses a tron paramagnetic resonance (EPR) measurements (1). single line absorber to investigate the energy levels of the Such comparisons for the naturally occuring heme group have 57Fe nuclei produced by decay of 57Co in the source (Fig. 2c). previously been impossible since heme cannot ordinarily Information can thus be gained regarding the iron complex be prepared in a five-coordinate state and heme in solution that exists during times of order 0.1 usec after electron cap- is oxidized to hemin upon exposure to oxygen. ture (12, 13). We have used Mdssbauer emission spectroscopy to study Although the absorption technique has received more an oxygenated heme complex, produced in frozen solution attention, M6ssbauer emission spectroscopy can offer distinct by nuclear decay from the isomorphous 57Co-labeledd com- advantages for certain biological studies. It requires far less pound (Fig. 1). Our results are compared with those obtained material than the absorption method if cobalt of high specific from Mbssbauer absorption spectroscopy of oxyhemoglobin.§ activity is used. (Less than 1 nmol of 57Co can be sufficient.) We find no evidence that the iron-oxygen linkage in oxy- Moreover, it provides the unique opportunity of investigating hemoglobin is influenced by the stereochemical constraints some iron compounds that cannot be made by direct chemical imposed by the protein. methods. The emission spectra of their cobalt analogs can convey information about the desired iron compounds. Prep- Abbreviations: heme, Fe(II)-protoporphyrin IX; CoP, Co(II)- aration of a suitable 57Co complex does not assure, however, protoporphyrin IX; heme-(OMe)2, dimethyl ester of heme; CoP- that the desired iron compound can be studied, because the (OMe)2 dimethyl ester of CoP; EPR, electron paramagnetic resonance; oxy-, indicates complex with molecular oxygen. § The Mossbauer spectra of oxyhemoglobin are not well under- t Although CoP and heme are the prosthetic groups in the pro- stood in terms of electronic structure. Several theories have been teins, our solution studies used the more soluble CoP-(OMe)2 and advanced to account for the diamagnetism of the heme complex heme-(OMe)2. Esterification of the propionic acid groups of a me- (5-7) and the temperature dependence of the quadrupole splitting talloprotoporphyrin IX does not significantly influence the por- (8, 9). No model has been conclusively proven correct, although phyrin ring. recent evidence appears to favor an Fe(III)-02- complex (10, 11). 2396 Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Oxygenated Heme in Solution 2397

SOURCE ABSORBER 57Co (270 d) SOURCE ABSORBER

ELECTRON r CAPTURE r-

136 s

14.4 keV i_ 57Fe ABSORPTION SPECTROSCOPY EMISSION SPECTROSCOPY (a) (b) (c) FIG. 2. (a) Decay scheme of "7Co, showing only those transitions relevant to Mossbauer spectroscopy. (b) Schematic representation of Mossbauer absorption spectroscopy, showing emission of photons in the source and resonant absorption in the absorber. The quadrupole splitting of the 14.4-keV excited nuclear level in the absorber is measured. The isomer shift is not considered in the figure. (c) Schematic representation of Mossbauer emission spectroscopy. The quadrupole splitting of the excited nuclear level in the source is measured.

electron shell must rearrange in the aftermath of the nuclear purchased from New England Nuclear in the form of CoCl2 decay process. This rearrangement process leads to ejection in acidified solution (Lot 7712, 33 mCi/ml). All other chem- of Auger electrons, and highly charged positive iron ions icals were Fisher reagent grade. Cobalt chloride was dried can occur. The resulting Coulombic forces may disrupt the under vacuum and redissolved in dimethylformamide (10 chemical bonds and fragment the molecule. However, 57Co mCi/ml). Pyridine was distilled from KOH, and toluene from complexes involving large conjugated ring structures resist CaH2, before use. Dimethylformamide was treated with disruption. In cobalt phthalocyanine and vitamin B12 com- KOH and CaO and then vacuum-distilled. 57Co-enriched pounds there is no evidence for fragmentation and well-de- CoP-(OMe)2 (100 Ci/mol) was prepared by a modification fined spectra are observed (14, 15). Recently, 57Co-labeled of the method of Adler (18). Co(II) pyridine complexes have been studied by M6ssbauer A solution of CoP-(O0Me)2(C5H5N)2 was prepared by addition emission spectroscopy, and the results are in good agreement of 0.2 ml degassed pyridine to CoP-(OMe)2 and further degas- with those obtained by absorption studies of the correspond- sing the solution by the freeze-thaw method. An EPR spectrum ing Fe(IJ) compounds (16, 17). We have extended these in- showed that no other paramagnetic cobalt species was vestigations to the porphyrin system by comparing the pa- present, and, in particular, that there was no oxyCoP-(ONle)r2 rameters of heme-(OMe)2(C5H5N)2 obtained from Mossbauer (C5H5N). The CoP-(OMe)2(C5H5N)2 solution was syringed into absorption and emission spectroscopy. a nylon M6ssbauer sample cell (0.5-inch diameter) under a nitrogen atmosphere. EXPERIMENTAL OxyCoP-(OMe)2(C5H5N) was prepared from the bispyridine Materials complexI. The filled M6ssbauer sample cell was placed in a Protoporphyrin IX dimethyl ester, Grade 1, was purchased Schlenk tube that was in contact with a dry ice-acetone bath. from Sigma and used without further purification. 57Co was Oxygen was passed through the tube for 1 hr while the solution was agitated mildly. The Schlenk tube was then immersed in liquid nitrogen, and the cell containing the frozen sample was transferred rapidly to the M6ssbauer cryostat. Before the M6ssbauer experiment, EPR measurements 0 showed that the material labeled with 57CoP-(OMe)2(C5H5N)2 could be completely and reversibly oxygenated. However, complete oxygenation was not achieved in the 1Idssbauer F (a) cell. This is believed to be due to the differences in the oxygenation and freezing processes caused by differences in sample volume and sample containers. I Methods 0~ A constant-acceleration Mossbauer spectrometer was used C') (19). The M6ssbauer source, the 57Co-labeled porphyrin com- m pound, was mounted in the tail section of a Janis variable- temperature cryostat. The y-rays could pass horizontally through i\Iylar windows. The drive unit was mounted out- side the cryostat. The drive rod terminated in a ring through which a Xe-CO2 proportional counter was positioned. A ABSORBER VELOCITY IN mm/sec K4Fe(CN)6 3H20 absorber (0.12 mg 57Fe/cm2) was mounted FIG. 3. (a) Mossbauer emission spectrum of CoP-(OMe)2- on the ring in front of the counter window. The temperature (C5H5N)2 at 4.2OK. The solid line is the result of fitting two doub- of the source could be stabilized anywhere between 4.20K lets to the data by the method of least squares. (b) Mossbauer and room temperature. The counting rates ranged from 200- emission spectrum of the oxygenated sample measured at 4.20K. The solid line is the result of fitting three doublets to the data. ¶ Since the EPR spectra of oxyCoP-(OMe)2(C5HrN) and oxy- The bracket designates the doublet assigned to oxyCoP-(OMe)2- CoP-(OMe)2(Im) are extremely similar, the use of pyridine rather (C5H6N). The absorber in both experiments was K4Fe(CN)6 * 3H20 than imidazole should have no significant effect on our conclu- at room temperature. sions. Downloaded by guest on September 25, 2021 2398 Biochemistry: Marchant et al. Proc. Nat. Acad. Sci. USA 69 (1972) TABLE 1. Quadrupole splittings, AEQ, and isomer shifts, 6, for heme-(OWe)2 complexes Temperature Complex Method (OK) AEQ (mm/sec) b(mm/sec)* Heme.(OMe)2(C5H5N)2 Emission 4.2 1.25 4± 0.03 0.47 i 0.02 Heme-(OMe)2(C5H5N)2 Absorption 4.2 1.12 ± 0.02 0.45 i 0.02 Oxyheme-(OMe)2(C5H5N) Emission 4.2 2.28 =i4 0.04 0.27 + 0.02 0.05 83 91 + 0.08 0.30 4± - 0.12 131 1 99 + 0.08 0.25 -t 0.05 -0.12 172 1.94 + 0.08 0.22 ± 0.05 -0.12

* Isomer shifts are quoted relative to metallic iron. The definition of 8 is the same as that used in Mossbauer absorption spectroscopy; i.e., a > 0 corresponds to a transition energy greater than that in metallic iron.

2000 counts/sec in the 14.4-keV line, depending upon the sec, may well be due to high-spin ferrous iron. This absorp- source used. The system was calibrated with a stationary tion was present in different intensities in different prepara- 57Co(Cu) source and a sodium nitroprusside absorber mounted tions. We therefore attribute it to a chemical impurity rather on the ring. EPR spectra were taken at 770K in a Varian than to a species resulting from disruption of the molecule 4500 EPR spectrometer. after nuclear decay. This impurity would have to contain Co in a state not readily observable by EPR, since this technique RESULTS gave no evidence that any species other than the bispyridine Me2CoP(C5H5N)2 complex was present. Fig. 3a shows the Mo6ssbauer emission spectrum of the heme- To test the similarity of the "'Co decay product to the (OMe)2(C5H5N)2 compound resulting from the decay of 57CoP- corresponding iron compound, we did a conventional iMss- (OMe)2(C5H5N)2, measured at 4.20K. A well-defined doublet bauer absorption experiment with Fe(II)-protoporphyrin and some additional absorption can be seen. The spectrum IX dimethyl ester [heme-(OMe)2] dissolved in alkaline aqueous was fitted by the least squares method with two doublets of pyridine (pyridine-0.1 N aqueous NaOH, 1:1, v/v). The Lorentzian line-shape. Within a doublet, the two lines were compound was reduced by the addition of excess Na2S204. constrained to have equal areas; the other fitting parameters While this was not the exact solvent system used for the were free. The prominent doublet, containing about 70% "7Co complex, it was the most similar one feasible. The spec- of the total absorption, showed a quadrupole splitting AEQ trum at 4.20K showed an isomer shift that agrees within = 1.25 mm/sec and an isomer shift 0.47 mm/sec (relative the experimental uncertainty with that found for CoP-(OMe)2- to iron metal). These parameters are indicative of Fe in the (CXH5N)2 and a quadrupole splitting about 10% smaller low-spin ferrous state, and we therefore assign the spectrum to than that for CoP-(OMe)2(C5H5N)2 (Table 1). It is difficult to heme-(OMe)2(C5H5N)2. The lines of this doublet are appreci- assess the significance of this rather small deviation, since ably narrower (0.38 mm/sec full width at half maximum) the two compounds were in different solvent systems. Con- than those seen in 57Co-labeled cobalt phthalocyanine ( 0.55 sidering the range of quadrupole splittings observed in low- mm/sec) and vitamin B12 compounds (0.9-1.0 mm/sec) (14, spin ferrous hemes [AEQ = 0.65 mm/sec in bispyridine meso- 20). The additional absorption, represented in the least heme dimethyl ester; AEQ = 1.08 mm/sec in bispyridine squares fit by lines at about +0.55 mm/sec and -1.75 mm/ 2,4-diacetyl deuteroheme dimethyl ester (21)], the discrep- ancy in AEQ does not appear to be very significant. OxyCoP-(OMe)2(C5H5N) Fig. 3b shows the result of oxygenating the sample used for 2.3- I~~~~~~~c recording the spectrum shown in Fig. 3a. The M6ssbauer AEQ 2.2- spectrum has changed drastically; two new lines have ap- fmml to the total Fs]ec 2.1- peared. The strength of this doublet relative emission spectrum of the sample varied with the thoroughness .9- of the oxygenation procedure. Fig. 3b represents our best 1.8- result. o oxyheme-(OMe)2(C5H5N) was fitted with three doublets. Since one of .7- x oxyhemoglobin The spectrum the minor components may be ferric, showing magnetic splitting, this six-line array may not be strictly justified. Although o 50 loo 160 200 T [OK] a good fit to the data was obtained, its quality was not very some This FIG. 4. Quadrupole splitting AEQ against temperature for sensitive to the values of parameters. insensitivity is a consequence of fitting data of low counting statistics oxyheme-(OMe)2(C5H5N) and oxyhemoglobin. Values for. oxy- fit hemoglobin are taken from absorption experiments by Lang and with a large number of free parameters. However, the Marshall (7). was fairly sensitive to the positions of the lines in the major Downloaded by guest on September 25, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Oxygenated Heme in Solution 2399

doublet, giving AEQ = 2.28 mm/sec and a = 0.27 mm/sec We thank Drs. P. G. Debrunner and H. Frauenfelder for their support and for suggestions in preparing this manuscript, Dr. J. L. at 4.20K. The uncertainties quoted are more than twice the Groves for discussion and assistance in evaluating the data, and standard deviations that resulted from the least squares Mr. Everett Knox for technical assistance. This work was sup- procedure. The positions of the other four lines are much less ported in part by National Institutes of Health Grant HL 13531 reliable, but agree with those fitted for the bispyridine sample and U.S. Public Health Service Grant GM 16406. L. M. is a Na- (Fig. 3a). tional Institutes of Health Predoctoral Fellow. B. M. H. is an The values of AEQ and a for the major doublet in Fig. 3b Alfred P. Sloan Fellow. agree within uncertainties with those published for oxyhemo- 1. Hoffman, B. M. & Petering, D. H. (1970) "Coboglobins: globin (7). To further check the similarity of the iron site Oxygen-Carrying Cobalt-Reconstituted Hemoglobin and in our oxyheme compound to that in oxyhemoglobin, we Myoglobin," Proc. Nat. Acad. Sci. USA 67, 637-643. 2. Stynes, H. C. & Ibers, J. A. (1972) "Thermodynamics of the have measured the temperature dependence of AEQ (Table Reversible Oxygenation of Amine Complexes of Cobalt(II) 1). Fig. 4 shows that, within the uncertainties, agreement Protoporphyrin IX Dimethyl Ester in a Nonaqueous Me- is good. In the spectra taken at temperatures above 4.20K, dium," J. Amer. Chem. Soc. 94, 1559-1562. the unknown temperature dependence of the minor lines 3. Hoffman, B. M., Spilburg, C. A. & Petering, D. H. (1971) made less reliable. The quadrupole split- "Coboglobins: Cobalt Substitution and the Nature of the least squares fitting Prosthetic Group-Apoprotein Interaction in Hemoglobin tings of the major doublet were therefore estimated from the and Myoglobin," Cold Spring Harbor Symp. Quant. Biol. apparent positions of the lines. The asymmetric uncertainties XXXVI, 343-348. quoted in Table 1 reflect the fact that outer bounds for the 4. Spilburg, C. A. & Hoffman, B. M. (1972) "Coboglobins: line positions could be determined more reliably than inner Influence of Apo-Myoglobin on Oxygen Binding to Cobalt Protoporphyrin IX," J. Biol. Chem. in press. limits. 5. Weiss, J. J. (1964)"Nature of the Iron-Oxygen Bond in Oxy- DISCUSSION haemoglobin," Nature 202, 83-84. 6. Pauling, L. (1964) "Nature of the Iron-Oxygen Bond in Oxy- From comparison of the parameters for heme-(OMe)2(C5H5N)2 haemoglobin," Nature 203, 182-183. as obtained by M6ssbauer emission and absorption spectros- 7. Lang, G. & Marshall, W. (1966) "Mbssbauer effect in some we the 57Co haemoglobin compounds," Proc. Phys. Soc. London 87, 3-34. copy, conclude that decay 57Fe and the sub- 8. Eicher, H. & Trautwein, A. (1969) "Electronic Structure and sequent electronic rearrangement do not cause measurable Quadrupole Splittings of Ferrous Iron in Hemoglobin," J. fragmentation of the porphyrin ring or loss of axial pyridine Chem. Phys. 50, 2540-2551. ligands. 9. Trautwein, A., Eicher, H., Mayer, A., Alfsen, A., Waks, M., As verified by EPR spectroscopy, oxygenation of 57Co-labeled Rosa, J. & Beuzard, Y. (1970) "Modification of the Elec- tronic Structure of Ferrous Iron in Hemoglobin by Liganda- CoP-(OMe)2(C5H5N)2 produces oxyCoP-(OMe)2(C5H5N). tion and by Alterations of the Protein Structure Inferred We have shown that the Mdssbauer parameters of the daugh- from M6ssbauer Measurements," J. Chem. Phys. 53,963-967. ter compound of oxy 57CoP-(OMe)2(C5H5N) agree well with the 10. Wittenberg, J. B., Wittenberg, B. A., Peisach, J. & Blum- values for oxyhemoglobin. The fact that these distinctive berg, W. E. (1970) "On the State of the Iron and the Nature M6ssbauer can for a heme complex of the Ligand in Oxyhemoglobin." Proc. Nat. Acad. Sci. parameters be obtained USA 67, 1846-1853. in solution suggests that the electronic structure of the heme 11. Koster, A. S. (1972) "Electronic State of Iron in Hemoglo- iron in oxyhemoglobin is not measurably influenced by the bin, Myoglobin, and Derivatives, as Inferred from X-Ray proteinl1. This parallels the conclusion from EPR studies that Fluorescence Spectra," J. Chem. Phys. 56, 3161-3164. the electronic structure of oxyCoP is not changed by incor- 12. Wertheim, G. K. (1961) "M6ssbauer Effect: Applications to Magnetism," J. Appl. Phys. S32, 110-117. poration into a protein (1). Thus, the greater oxygen affinity 13. Wertheim, G. K., Kingston, W. R. & Herber, R. H. (1962) of the heme prosthetic group in hemoglobin does not show "M6ssbauer Effect in Iron(III) Acetylacetonate and Chem- itself as a 'perturbation of the electronic state of the iron as ical Consequences of K Capture in Cobalt(III) Acetylace- measured by the M6ssbauer parameters and is, therefore, tonate," J. Chem. Phys. 37, 687-690. not ascribable to protein-induced changes of the 14. Mullen, R. T. (1970) in Mbssbauer Effect Mehodology Vol. 5, probably ed. Gruverman, I. J. (Plenum Press, New York), pp. 95- electronic state of iron in oxyheme. Comparison of the M6ss- 105. bauer parameters of deoxyhemoglobin with those of the cor- 15. Nath, A., Klein, M. P., Kundig, W. & Lichtenstein, D. responding five-coordinate heme in solution could give addi- (1970) " M6ssbauer Studies of After-Effects of Auger Ioniza- tional information about the mechanism of oxygen binding**. tion Following Electron Capture in Cobalt Complexes," Radiat. Eff. 2, 211-234. The measurements reported here give evidence that M6ss- 16. Saito, N., Takeda, M. & Tominaga, T. (1971) "M6ssbauer bauer emission spectroscopy is suitable for the study of heme Spectroscopic Study of 67Co-Labeled Cobalt Pyridine Com- complexes. The inherently greater sensitivity of this technique plexes," Radiochem. Radioanal. Lett. 6, 169-175. could be exploited in the investigation of single crystals of 17. Friedt, J. M., Poinsot, R. & Sanchez, J. P. (1971) "M6ss- heme protein. Crystallized coboglobins (1) would be promis- bauer Emission Spectroscopy of Some 57Co-Labeled Cobalt Complexes," Radiochem. Radioanal. Lett. 7, 193-203. ing candidates for such work. 18. Adler, A. D., Longo, F. R., Kampas, F. & Kim, J. (1970)"On the preparation of metalloporphyrins," J. Inorg. Nucl. Chern. 32, 2443-2445. 11 We have observed a similar spectrum for oxygenated cyto- 19. Kankeleit, E. (1964) "Velocity Spectrometer for M6ssbauer chrome P45Oam. Furthermore, the Mossbauer spectrum in a high Experiments," Rev. Sci. Instrum. 35, 194-197. magnetic field shows that oxygenated P450cam, like 20. Nath, A., Harpold, M., Klein, M. P. & Kundig, W. (1968) oxyhemoglo- for Biologically Impor- bin, is (M. et in "Emission M6ssbauer Spectroscopy diamagnetic Sharrock, al., preparation). tant Molecules. Vitamin B12, Its Analogs, and Cobalt Phtha- Preliminary experiments on five-coordinate57CoP-(OMe)2- locyanine," Chem. Phys. Lett. 2, 471-476. (C6HrN) indicate that the Mdssbauer parameters of heme-(OMe2)- 21. Bearden, A. J., Moss, T. H., Caughey, W. S. & Beaudreau, (CbH5N) are significantly different (AEQ = 1.8 mm/sec and a C. A. (1965) "M4ssbauer Spectroscopy of Heme and Hemin 0.7 mm/sec at 4.20K) from those of deoxyhemoglobin (9). Compounds," Proc. Nat. Acad. Sci. USA 53, 1246-1253. Downloaded by guest on September 25, 2021