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The heme-regulated phosphodiesterase fromEscherichia coli(EcDOS), which is a heme redox-dependent enzyme, is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence ofEcDOS activity on the redox state. | iFEBS Journal Critical roles of Leu99 and Leu115 at the heme distal side in auto-oxidation and the redox potential of a heme-regulated phosphodiesterase from Escherichia coli Nao Yokota Yasuyuki Araki Hirofumi Kurokawa Osamu Ito Jotaro Igarashi and Toru Shimizu Institute of Multidisciplinary Research for Advanced Materials Tohoku University Sendai Japan Keywords auto-oxidation CO binding heme-sensor protein O2 binding phosphodiesterase Correspondence T. Shimizu Institute of Multidisciplinary Research for Advanced Materials Tohoku University 2-1-1 Katahira Aoba-ku Sendai 980-8577 Japan Fax 81 22 217 5604 5390 Tel 81 22 217 5604 5605 E-mail shimizu@tagen.tohoku.ac.jp Received 13 December 2005 revised 14 January 2006 accepted 18 January 2006 doi 10.1111 j.1742-4658.2006.05145.x The heme-regulated phosphodiesterase from Escherichia coli Ec DOS which is a heme redox-dependent enzyme is active with a ferrous heme but inactive with a ferric heme. Global structural changes including axial ligand switching and a change in the rigidity of the FG loop accompanying the heme redox change may be related to the dependence of Ec DOS activity on the redox state. Axial ligands such as CO NO and O2 act as inhibitors of Ec DOS because they interact with the ferrous heme complex. The X-ray crystal structure of the isolated heme-bound domain Ec DosH shows that Leu99 Phe113 and Leu115 indirectly and directly form a hydrophobic triad on the heme plane and that they should be located at or near the ligand access channel of the heme iron. We generated L99T L99F L115T and L115F mutants of Ec DosH and examined their physicochemical characteristics including auto-oxidation rates O2 and CO binding kinetics and redox potentials. The Fe III complex of the L115F mutant was unstable and had a Soret absorption spectrum located 5 nm lower than those of the wild-type and other mutants. Auto-oxidation rates of the mutants 0.049-0.33 min-1 were much higher than that of the wildtype 0.0063 min-1 . .
