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 spectros- copy 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 spectros- copy 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 reso- nance; 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 solu- tion 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 oxy- genation and freezing processes caused by differences in sam- ple 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.