Iron L-Edge X-ray Absorption Spectroscopy of Myoglobin Complexes and Photolysis Products Hongxin Wang, ² Gang Peng, ‡ Lisa M. Miller, ²,§ Eva M. Scheuring, § S. J. George, ‡ Mark R. Chance,* ,§ and Stephen P. Cramer* ,²,‡ Contribution from the Energy and EnVironment DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720, Department of Applied Science, UniVersity of California, DaVis, California 95616, and Department of Physiology and Biophysics, Albert Einstein College of Medicine of YeshiVa UniVersity, Bronx, New York 10461 ReceiVed May 1, 1996 X Abstract: We demonstrate the first application of L-edge X-ray absorption spectroscopy (XAS) to the electronic characterization of biological photolysis products. The experimental L-edge XAS spectra of deoxymyoglobin (deoxy Mb), oxymyoglobin (MbO 2 ), carbonmonoxymyoglobin (MbCO), and the low-temperature photoproducts (Mb*CO and Mb*O 2 ) are presented and compared to simulated spectra using a ligand field multiplet calculation. This analysis indicates that MbCO and MbO 2 are both low spin and does not support some previous studies which suggest that MbO 2 has an intermediate spin. Both photoproducts, Mb*CO and Mb*O 2 , are different from deoxy Mb in the Fe II electronic structure. In addition, different low-temperature photolysis intermediates are suggested for MbCO and MbO 2 . The L-edge XAS spectra for Fe III in aquometmyoglobin (met Mb) and azidomet myoglobin (MbN 3 ) provide a comparison of the ferrous versus ferric myoglobin species. Finally, the special advantages of using soft X-ray absorption spectroscopy for understanding the electronic transitions coupled to photolysis-induced structural changes are discussed. Introduction The process of photodissociation in which bonds are broken following absorption of a photon is of fundamental importance in biology and chemistry. 1-3 Photochemically generated in- termediates can initiate subsequent reactions and produce special products for various scientific and industrial applications. 1 X-ray absorption spectroscopy (XAS) has been used to char- acterize a number of photochemically generated samples which contain metal centers, especially in the area of biophysics. In one of the first solution studies, Mills et al. 4-7 used a 20-Hz repetition rate Nd:YAG laser to photolyze carbonmonoxymyo- globin (MbCO), and they monitored the photolysis product (Mb*CO) with time-resolved K-edge XAS. Since then, other systems for time-resolved K-edge XANES and EXAFS have been used by Clozza et al., 8 by Thiel et al., 9 and by Chance et al. 10 on flowing solution samples. K-edge XAS without rapid time resolution has also been used to study various trapped intermediate photoproducts such as low-temperature myoglobin and hemoglobin photoproducts, 11-13 and the S states in photo- system II. 14 Meanwhile, time-resolved K-edge XAS has been used in various fields to characterize the changes in gas-phase plasmas, 15 ablated particles, 16 doped glasses, 17 and solid state material samples 18 following laser irradiation. To date, all XAS applications on biological photoproducts have only involved the use of hard X-ray radiation (>2000 eV). L-edge XAS of the first row (3d) transition metals, which utilizes soft X-rays in the energy region of 500-1000 eV, has several advantages over K-edge XAS. 19 Generally, soft X-ray XAS has 3-5 times better energy resolution, resulting in sharper spectral features. Transitions at the L edge (2p f 3d) are dipole-allowed and provide spectra that are more intense and structured than the dipole-forbidden K-edge (1s f 3d) transi- tions. This is especially valuable for studying ligand association and dissociation in metalloproteins, such as hemeproteins, where the metal (3d) orbitals are the ligand-bonding orbitals. In addition, L-edge XAS spectra can be interpreted by ligand field calculations, 20-23 which provide a more detailed understanding of metalloprotein electronic structures. L-edge XAS is sensitive * Address correspondence to these authors. ² Lawrence Berkeley National Laboratory. ‡ University of California. § Albert Einstein College of Medicine of Yeshiva University. X Abstract published in AdVance ACS Abstracts, March 1, 1997. (1) Bershon, R. In Molecular Photodissociation Dynamics; Ashford, M. N. R., Baggot, J. E., Eds.; Royal Society of Chemistry: London, 1987; p 61. (2) Wang, H.; Chen, X.; Weiner, B. R. J. Phys. Chem. 1993, 97, 12261. (3) Chen, X.; Wang, H.; Weiner, B. R.; Hawley, M.; Nelson, H. H. J. Phys. Chem. 1993, 97, 12269. (4) Mills, D. M. Phys. Today 1984, 4, 22. (5) Mills, D. M.; Pollock, V.; Lewis, A.; Harootian, A.; Huang, J. Nucl. Instrum. 1984, 222, 351. (6) Mills, D. M.; Lewis, A.; Harootian, A.; Huang, J.; Smith, B. Science 1984, 223, 811. (7) Pollock, V.; Mills, D. M. Nucl. Instrum. Methods 1984, A226, 668. (8) Clozza, A.; Castellano, A. C.; Longa, S. D.; Giovannelli, A.; Bianconi, A. ReV. Sci. Instrum. 1989, 60, 2519. (9) Thiel, D. J.; Livins, P.; Stern, E. A.; Lewis, A. Nature 1993, 362, 40. (10) Chance, M. R.; Wirt, M. D.; Scheuring, E. M.; Miller, L. M.; Xie, A.; Sidelinger, D. ReV. Sci. Instrum. 1993, 64, 2035. (11) Chance, B.; Fischetti, R.; Powers, L. Biochemistry 1983, 22, 3820. (12) Powers, L.; Chance, B.; Chance, M. R.; Campbell, B.; Friedman, J.; Khalid, S.; Kumar, C.; Naqui, A.; Reddy, K. S.; Zhou, Y. Biochemistry 1987, 26, 4785. (13) Chance, M. R.; Miller, L. M.; Fischetti, R. F.; Scheuring, E.; Huang, W. X. Sclavi, B.; Hai, Y.; Sullivan, M. Biochemistry 1996, 35, 9014. (14) Yachandra, V. K.; Guiles, R. D.; McDermott, A. E.; Cole, J. L.; Zimmermann, V. L. J.; Sauer, K.; Klein, M. K. Phys. B 1989, 158, 78. (15) Wang, L. S.; Lin, Z. Q.; Zhang, H. H.; He, X. F. Acta Phys. Sin. (OVerseas Ed.) 1994, 3, 909. (16) Ohyanagi, T.; Miyashita, A.; Murakami, K. J. Appl. Phys., Part I 1994, 33, 2586. (17) Lee, J. M.; Paesler, M. A.; Sayers, D. E.; Fontaine, A. Phys. B 1989, 158, 52. (18) Kassner, M. E.; Li, X.; McQueen, H. J. Mater. Sci. Eng. 1993, A169, 9. (19) Cramer, S. P.; deGroot, F. M. K.; Ma, Y.; Chen, C. T.; Sette, F.; Kipke, C. A.; Eichhorn, D. M.; Chan, M. K.; Armstrong, W. H.; Libby, E.; Christou, G.; Brooker, S.; McKee, V.; Mullins, O. C.; Fuggle, J. C. J. Am. Chem. Soc. 1991, 113, 7937. 4921 J. Am. Chem. Soc. 1997, 119, 4921-4928 S0002-7863(96)01446-1 CCC: $14.00 © 1997 American Chemical Society