Using X-ray absorption spectroscopy, we investigated the active site of horseradish peroxidase (HRP) compound II at two different pH values. The results indicate that the bond length of the sixth coordinated ligand of the active site was 1.90 +/- 0.02 A at pH 7, decreasing to 1.72 +/- 0.02 A at pH 10. The average iron-to-pyrrole nitrogen and the proximal ligand bond lengths showed no significant changes. The position of higher coordination shells around the iron center changed, implying that some movement or deformation of nearby amino acid residues and/or of the heme occurred. Results of this study suggest that the decrease of the Fe-O bond length of HRP compound II at the higher pH might be attributed to the loss of a hydrogen bond which is present between the oxygen ligand and an amino acid residue in the heme pocket at pH 7.

Native lactoperoxidase, compound III, and the reduced forms (at pH 6 and 9) were studied using X-ray absorption spectroscopy (XAS). Native lactoperoxidase has four pyrrole nitrogen ligands at an average distance of 2.04 +/- 0.01 A, a proximal ligand at 1.91 +/- 0.02 A, and a sixth (distal) ligand at 2.16 +/- 0.03 A. Lactoperoxidase native enzyme has a first coordination shell structure that is similar to that of native lignin peroxidase [Sinclair, R., Yamazaki, I., Bumpus, J., Brock, B., Chang, C.-S., Albo, A., & Powers, L. (1992) Biochemistry 31, 4892-4900] and different from that of horseradish peroxidase [Chance, B., Powers, L., Ching, Y., Poulos, T., Schonbaum, G., Yamazaki, I., & Paul, K. (1984) Arch. Biochem. Biophys. 235, 596-611]. Similarly, lactoperoxidase compound III resembles lignin peroxidase compound III. The five-coordinated ferrous form was stable at pH 9, but at pH 6 it was rapidly converted to the six-coordinated form with a distal ligand at 2.18 +/- 0.03 A. No evidence typical of changes in spin state was obtained at the different pH values.

Elementary steps or geminate states in the reaction of gaseous ligands with transport proteins delineate the trajectory of the ligand and its rebinding to the heme. By use of kinetic studies of the 765-nm optical "conformation" band, three geminate states were identified for temperatures less than approximately 100 K. MbCO, which is accumulated by photolysis between 1.2 and approximately 10 K, was characterized by our previous optical and X-ray absorption studies [Chance, B., Fischetti, R., & Powers, L. (1983) Biochemistry 22, 3820-3829]. Between 10 and approximately 100 K, geminate states that are also identified that have recombination rates of approximately 10(3) s-1 and approximately 10(-5) s-1 (40 K). Thus, it is possible to maintain a steady-state nearly homogeneous population of the slowest recombining geminate state, Mb, by regulated continuous illumination (optical pumping). Both X-ray absorption and resonance Raman studies under similar conditions of optical pumping show that the heme structure around the iron in Mb is similar to that of MbCO. In both geminate states, the iron-proximal histidine distance remains unchanged (+/- 0.02 A) from that of MbCO while the iron to pyrrole nitrogen average distance has not fully relaxed to that of the deoxy state. In MbCO the CO remains close to iron but not bound, and the Fe...CO angle, which is bent in MbCO (127 +/- 4 degrees C), is decreased by approximately 15 degrees [Powers, L., Sessler, J. L., Woolery, G. L., & Chance, B. (1984) Biochemistry 23, 5519-5523]. The CO molecule in Mb, however, has moved approximately 0.7 A further from iron. Computer graphics modeling of the crystal structure of MbCO places the CO in a crevice in the heme pocket that is just large enough for the CO molecule end-on. Above approximately 100 K resonance Raman studies show that this structure relaxes to the deoxy state.