Diatomic Ligand Discrimination by the Heme Oxygenases from Neisseria meningitidis and Pseudomonas aeruginosa

Abstract

Heme oxygenases have an increased binding affinity for O 2 relative to CO. Such discrimination is critical to the function of HO enzymes because one of the main products of heme catabolism is CO. Kinetic studies of mammalian and bacterial HO proteins reveal a significant decrease in the dissociation rate of O 2 relative to other heme proteins such as myoglobin. Here we report the kinetic rate constants for the binding of O 2 and CO by the heme oxygenases from Neisseria meningitidis (nmHO) and Pseudomonas aeruginosa (paHO). A combination of stoppedflow kinetic and laser flash photolysis experiments reveal that nmHO and paHO both maintain a similar degree of ligand discrimination as mammalian HO-1 and the HO from Corynebacterium diphtheriae. However, in addition to the observed decrease in dissociation rate for O 2 by both nmHO and paHO, kinetic analyses show an increase in dissociation rate for CO by these two enzymes. The crystal structures of nmHO and paHO both contain significant differences from the mammalian HO-1 and bacterial C. diphtheriae HO structures, which suggests a structural basis for ligand discrimination in nmHO and paHO. Heme oxygenase (HO)2 catalyzes the oxidative degradation of heme to biliverdin, iron, and CO (Fig. 1). HO has been identified in a wide array of organisms, including mammals (1, 2), insects (3, 4), and photosynthetic organisms (5, 6). Of particular interest, HO is present in many pathogenic bacteria (7-10), including Neisseria meningitidis (11) and Pseudomonas aeruginosa (12). These pathogenic bacteria have developed sophisticated heme uptake systems that harness the iron from hemecontaining proteins present in the host (13-15). The HO enzymes from N. meningitidis (nmHO) and P. aeruginosa (paHO) are both essential for the utilization of iron from imported heme, and the crystal structures of these two bacterial HO enzymes have now been solved (16,17). Although most HOs hydroxylate exclusively the ␣-meso heme carbon (Fig. 1), paHO is unusual because the ␥-meso carbon is the predominate site of hydroxylation (18) even though the structure paHO is very much the same as other HOs. The difference in hydroxylation patterns is due to an ϳ100 o rotation of the heme in paHO relative to other HOs which places the ␥-meso carbon at the same position in the active site as the ␣-meso carbon in other HOs (16).The Fe(II) atom of the heme prosthetic group of heme proteins is an efficient binder of O 2 , NO, and CO, and the binding by heme proteins of these diatomic molecules is of critical importance to physiological processes such as respiration, vasodilation, and neurotransmission (19 -21). A common feature shared by all heme proteins is the need to not only bind its target ligand but also to discriminate against the binding of heme ligands of similar size and shape. Fe(II) adducts of CO are normally linear because backbonding is optimized by the overlap of Fe(II) d-orbitals with the empty * orbitals of . In contrast, Fe(II)-NO and Fe(II)-O 2 are naturally bent in order to maximize overlap with ...

Discrimination between CO and O2 in Heme Oxygenase: Comparison of Static Structures and Dynamic Conformation Changes following CO Photolysis

“…The conformational change upon CO binding is not restricted to mammalian HO-1. A similar change has been reported in one bacterial HO homolog (17) but was absent in another (18). Steric hindrance thus seems to be one of the mechanisms for discrimination between CO and O 2 in HO.…”

Ruffling of Metalloporphyrins Bound to IsdG and IsdI, Two Heme-degrading Enzymes in Staphylococcus aureus

“…The regiospecificity is proposed to be dependent on steering and shielding by the water network on the distal side (40). An exception is an HO from Pseudomonas aeruginosa, which favors the ␦-meso carbon through a reorientation of the porphyrin ring in the active site (41). In P. aeruginosa, a bacterial phytochrome is implicated in HO product release (42).…”

Binding of O2 and NO to Heme in Heme-Nitric Oxide/Oxygen-Binding (H-NOX) Proteins. A Theoretical Study

“…6–12 Some of these bacterial proteins from eukaryotes and facultative aerobic prokaryotes also exclude O 2 while binding NO selectively, the same characteristic as sGC. These proteins may be called NO-selective H-NOXs.…”