The Hydroxide Complex of Pseudomonas aeruginosa Heme Oxygenase as a Model of the Low-Spin Iron(III) Hydroperoxide Intermediate in Heme Catabolism: 13 C NMR Spectroscopic Studies Suggest the Active Participation of the Heme in Macrocycle Hydroxylation Gregori A. Caignan, ² Rahul Deshmukh, ‡ Yuhong Zeng, ² Angela Wilks, ‡ Richard A. Bunce, § and Mario Rivera* ,² Contribution from the Department of Chemistry, The UniVersity of Kansas, Lawrence, Kansas 66045-7582, Department of Pharmaceutical Sciences, School of Pharmacy, UniVersity of Maryland, Baltimore, Maryland 21201-1180, and Department of Chemistry, Oklahoma State UniVersity, Stillwater, Oklahoma 74078 Received May 14, 2003; E-mail: mrivera@ku.edu Abstract: 13 C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudo- monas aeruginosa heme oxygenase (Fe III -OH), where OH - has been used as a model of the OOH - ligand to gain insights regarding the elusive ferric hydroperoxide (Fe III -OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe III -OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S ) 1 /2 and S ) 3 /2 spin states, and the two less abundant populations exhibit pure, S ) 3 /2 and S ) 1 /2, (d xy) 1 electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH - , is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOH - in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation. Introduction The enzyme heme oxygenase (HO) is intimately involved in the catabolism of heme. In this process, HO catalyzes the electron- and dioxygen-dependent breakdown of heme to biliverdin, iron, and carbon monoxide. 1 The catalytic cycle of HO (Scheme 1) starts when the ferric enzyme is reduced by NADPH cytochrome P450 reductase to its ferrous form, followed by the coordination of O 2 , which leads to the formation of an oxyferrous complex (Fe II -O 2 ). The latter accepts a second electron from the reductase and is thereby transformed into the ferric hydroperoxy (Fe III -OOH) oxidizing species, 2 which adds a hydroxyl group to the R-meso carbon to form R-meso- hydroxyheme (Scheme 1). 3,4 Investigations of the reactivity of HO toward peroxides and alkyl peroxides led to the conclusion that heme hydroxylation does not proceed via the formation of a high-valence compound I-like species. 3 Rather, the terminal oxygen of the coordinated peroxide adds to a porphyrin meso carbon, which results in the formation of R-hydroxyheme. In fact, spectroscopic evidence supporting this conclusion was recently obtained by cryoreduction of the oxyferrous complex of HO to produce an intermediate, identified by EPR spectros- copy to be the Fe III -OOH complex, which upon warming is converted into the R-hydroxyheme complex. 5,6 The R-meso- hydroxyheme complex of HO undergoes a subsequent O 2 - dependent elimination of the hydroxylated R-meso carbon as CO, with the simultaneous formation of verdoheme (Scheme ² The University of Kansas. ‡ University of Maryland. § Oklahoma State University. (1) Tenhunen, R.; Marver, H. S.; Schmid, R. J. Biol. Chem. 1969, 244, 6388- 6394. (2) Yoshida, T.; Noguchi, M.; Kikuchi, G. J. Biol. Chem. 1980, 255, 4418- 4420. (3) Wilks, A.; Torpey, J.; Ortiz de Montellano, P. R. J. Biol. Chem. 1994, 269, 29553-29556. (4) Ortiz de Montellano, P. R.; Wilks, A. AdV. Inorg. Chem. 2000, 51, 359- 407. (5) Davydov, R.; Macdonald, I. D. G.; Makris, T. M.; Sligar, S. G.; Hoffman, B. M. J. Am. Chem. Soc. 1999, 121, 10654-10655. (6) Davydov, R.; Kofman, V.; Fujii, H.; Yoshida, T.; Ikeda-Saito, M.; Hoffman, B. M. J. Am. Chem. Soc. 2002, 124, 1798-1808. Published on Web 09/04/2003 11842 9 J. AM. CHEM. SOC. 2003, 125, 11842-11852 10.1021/ja036147i CCC: $25.00 © 2003 American Chemical Society