STUDIES ON FERRIC FORMS OF CYTOCHROME-P-450 AND CHLOROPEROXIDASE BY EXTENDED X-RAY ABSORPTION FINE-STRUCTURE - CHARACTERIZATION OF FE-N AND FE-S DISTANCES

Cytochrome P-450 and chloroperoxidase are Fe-heme proteins with similar spectroscopic properties, which catalyze respectively the hydroxylation and halogenation of organic substances. The nature of and distances to the nonporphyrin (axial) ligands in these proteins are clearly of importance in understanding their catalytic cycles on a molecular level. This paper reports the first use of extended X-ray absorption fine structure (EXAFS) spectroscopy to study the iron environment in the ferric resting states of these two enzymes. First, analysis methods were developed for model iron porphyrin systems. Least-squares curve fits to the EXAFS data, using empirical phase and amplitude functions, led to the determination of interatomic distances in Fe porphyrins of known structure; Fe-Np distances were determined to within ±0.007 Å and the Fe-Cαand Fe-X (where X=O, S. N. to better than ±0.025 Å. Second, visual comparison of the protein data with that for the models allowed classification of the chloroperoxidase as high spin (iron out of plane) and P-450-LM-2 as low spin (iron in plane). The data for both oxidized enzymes demonstrate the presence of an axial sulfur ligand. Finally, detailed curve fitting analysis of the EXAFS revealed that the chloroperoxidase distances were Fe-Np=2.05 Å, Fe-Cα=3.09 Å, and Fe-S=2.30 Å. These are strikingly similar to the corresponding distances found in FeIII(PPIXDME)(SC6H4-p-NO2). The distances from EXAFS analysis for P-450-LM-2 were 2.00, 3.07, and 2.19 Å for the Fe to NP, Cα, and axial S ligands, respectively. The use of EXAFS for determining accurate interatomic distances, atomic types, and coordination numbers in these types of iron-heme proteins is clearly demonstrated. © 1978, American Chemical Society. All rights reserved.