title: 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.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Newmyer
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: T M
authors.initials: TM
authors.lastname: Loehr
authors.forename: P R
authors.initials: PR
authors.lastname: Ortiz de Montellano
mesh_terms: D016367:Baculoviridae / Q000235:genetics; D052160:Benzothiazoles; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006139:Guaiacol / Q000378:metabolism; D006420:Hemeproteins / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000235:genetics* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D013451:Sulfonic Acids / Q000378:metabolism
chemical_list: D052160:Benzothiazoles; D006420:Hemeproteins; D007093:Imidazoles; D011994:Recombinant Proteins; D013451:Sulfonic Acids; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006639:Histidine; D006139:Guaiacol; C029899:imidazole; D006861:Hydrogen Peroxide; D007501:Iron; D006735:Horseradish Peroxidase