Catalase HPII of Escherichia coli catalyzes the conversion of protoheme to cis-heme d

Abstract

Catalase HPII from aerobically grown Escherichia coli normally contains heme d but cultures grown with poor or no aeration produce HPII containing a mixture of heme d and protoheme IX. The protoheme component of HPII from anaerobically grown cells is converted into heme d during treatment of the purified enzyme with hydrogen peroxide. It is concluded that heme d found in catalase HPII is formed by the cis-hydroxylation of protoheme in a reaction catalyzed by catalase HPII using hydrogen peroxide as a substrate. The distal His128 residue of HPII is absolutely required for the protoheme to heme d conversion. Two mutant enzymes, Ala128 and Asn128, are catalytically inactive and contain only protoheme, which is unaffected by hydrogen peroxide treatment. The Asn201 residue is not an absolute requirement for heme conversion. The mutant enzyme Ala201 contains predominantly heme d and is partially active. However, insertion of a histidyl residue to give the His201 enzyme interferes with the heme conversion reaction. This mutant form is isolated as a protoheme enzyme with limited activity, and a reversible conversion to a heme d-like species occurs in vitro in the presence of continuously generated hydrogen peroxide.

An Electrical Potential in the Access Channel of Catalases Enhances Catalysis

“…Subsequent expression and purification were carried out as described previously (15). Catalase, Protein, and Spectral Determination-Catalase activity was determined by the method of Rorth and Jensen (28) in a Gilson oxygraph equipped with a Clark electrode.…”

“…HPII from E. coli contains two post-translational modifications in the active center, including an oxidized, cis-spirolactone, heme d (13), and a covalent bond between the N ␦ of His 392 and the C ␤ of Tyr 415 , the proximal side fifth ligand of the heme (14). Both modifications are generated self-catalytically by the catalase and seem to require some degree of catalase activity (15). Three channels, the main channel oriented perpendicular to the plane of the heme, the lateral channel approaching in the plane of the heme, and a channel leading to the central cavity, connect the active site to the exterior of the enzyme, providing routes for substrate ingress and product egress.…”

“…1). His 128 is essential for catalysis in HPII (15), and the importance of Val 169 in constricting the narrowest, hydrophobic portion of the channel has been investigated in yeast CATA (7) and HPII (21), although without a definitive conclusion. The importance of the other residues further up the channel has not been studied, and this paper focuses on a number of these residues in the main channel of HPII.…”

Catalase: A repertoire of unusual features

“…While hemin is ubiquitous in a wide variety of electron transfer proteins and enzymes, catalases are unique in having two types of metalloporphyrins, heme b and heme d, the latter represents a metallochlorin in which a pyrrole ring is reduced (9). Infact, in large subunit enzymes such as HPII, heme b is initially bound during assembly and it is subsequently oxidized by the catalase itself during the early rounds of catalysis (10). One of the most intriguing features of heme in catalases is that it can exist in two orientations relative to the active site residues.…”

Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli

“…Another relevant difference from the small subunit catalases lies in the presence of a cis-hydroxy spirolactone heme d that is "flipped" 180" around the axis defined by the a-y meso carbon atoms, relative to the orientation of the heme b found in the smaller catalases (Murshudov et al, 1996). The oxidation of the heme in HPII is catalyzed by HPII itself, which uses H202 as substrate (Loewen et al, 1993). However, the modification occurs on the proximal side of ring I11 of the heme, at a significant distance from the catalytic residues, His 128 and Asn 201, and a satisfactory mechanism for the oxidation has yet to be proposed.…”