Rescue of the Horseradish Peroxidase His-170 → Ala Mutant Activity by Imidazole:  Importance of Proximal Ligand Tethering

Abstract

The proximal iron ligand in horseradish peroxidase (HRP) is His-170. The H170A mutant of polyhistidine-tagged HRP (hHRP) has been expressed in a baculovirus system and has been purified and characterized. At pH 7, the Soret maximum of the mutant is at 414 nm rather than 403 nm. Resonance Raman spectra indicate that the protein is primarily 6-coordinate low-spin in the ferric state with a band in the ferrous state at 212 cm-1 indicative of distal histidine coordination to the iron. Exogenous imidazole (Im) binds to the enzyme with Kd = 22 +/- 4 mM. Reaction of H170A hHRP with H2O2 does not give spectroscopically detectable compound I or compound II intermediates but results in gradual degradation of the heme group. Nevertheless, H170A hHRP is catalytically active, and its guaiacol and ABTS peroxidase activities are improved 260- and 125-fold, respectively, in the presence of saturating concentrations of Im. The Km for the stimulatory effect of Im is 24 mM for both guaiacol and ABTS. The pH profile of H170A hHRP differs from that of wild-type hHRP, but the differences are essentially eliminated by Im. The rate of formation of "compound I" for H170A hHRP, determined by steady state kinetic methods, is k1 = 16 M-1 s-1 without Im and k1 = 2.4 x 10(4) M-1 s-1 with Im. The corresponding rate for wild-type hHRP is k1 = 4.4 x 10(6) M-1 s-1. The results indicate that Im binds in the cavity created by the H170A mutation, coordinates to the heme iron atom, and restores a large part of the catalytic activity by rescuing the rate of compound I formation. However, this rescue of the catalytic activity by Im is possibly limited by coordination of the heme to the distal histidine (His-42) in the H170A mutant. Thus, a primary function of the proximal histidine is to tether the iron atom to disfavor sixth ligand binding, particularly coordination of the iron to the distal histidine. In addition, strong hydrogen bonding of the proximal ligand may be critical for facilitating O-O bond cleavage in the formation of compound I.

Studies on the specificity of unprocessed and mature forms of phytanoyl-CoA 2-hydroxylase and mutation of the iron binding ligands

“…by guest, on May 12, 2018 www.jlr.org Downloaded from metal ions) have shown that histidine-to-alanine mutations can be "rescued" using imidazole (37)(38)(39)(40)(41)(42). Thus, all PAHX histidine mutants (except for the insoluble H213A mutant) were assayed for phytanoyl-CoA conversion in the absence and presence of imidazole ( Table 2).…”

Assignment of the Heme Axial Ligand(s) for the Ferric Myoglobin (H93G) and Heme Oxygenase (H25A) Cavity Mutants as Oxygen Donors Using Magnetic Circular Dichroism

“…This approach was pioneered by Barrick (15) for sperm whale myoglobin (Mb) 1 and has since been accomplished for cytochrome c peroxidase (16), heme oxygenase (17), and horseradish peroxidase (18). Studies of cavity mutants to date have focused on the dynamics of binding unnatural ligands (19)(20)(21)(22) and the resulting effects on catalytic activity (18).…”

The Hydrogenase Cytochrome b Heme Ligands of Azotobacter vinelandii Are Required for Full H 2 Oxidation Capability

“…Imd has been used with several substituted heme proteins (e.g., horseradish peroxidase H170A [31], cytochrome c peroxidase H175G [24], a soluble version of heme oxygenase H25A [47], and soluble guanylate cyclase H105G [50]) to serve as an exogenous ligand and rescue heme binding. In each of these studies with purified proteins, the iron in the native protein is five-coordinate with a single histidine as the proximal ligand and Imd replaced this ligand, albeit to various degrees.…”