Dataset Information

Redox- and anion-linked protonation sites in horseradish peroxidase: analysis of distal haem pocket mutants.

ABSTRACT: We have investigated the effects of mutations at residues His-42, Arg-38 and Phe-41 in the distal haem pocket of horseradish peroxidase on the changes in protonation state that accompany redox- and ligand-linked changes to the haem group. The mutations H42L and R38L result in the loss of a characteristic pH dependency in the visible spectrum of the ferrous form and a diminished dependency of the midpoint redox potential of the haem group on pH. The results support the view that His-42, with its pK probably modulated by Arg-38, provides the protonation site on the reduced enzyme that is responsible for these pH dependencies. The mutations H42L and R38L also have major effects on the binding of cyanide to the haem. We have already reported that binding of cyanide to the ferrous forms of these mutants becomes too weak to be measurable [Meunier, Rodriguez-Lopez, Smith, Thorneley and Rich (1995) Biochemistry 34, 14687-14692]. The pH dependency of the rate constants for binding of cyanide to the oxidized form of H42L suggests that CN- is the kinetically active species, in contrast with wild-type horseradish peroxidase, where HCN is the binding form. For the R38L variant, the pH dependency of cyanide binding suggests that the pK of His-42 in the absence of Arg-38 is raised to 7.5-8, in the oxidized form. In contrast with these changes, the mutant F41A exhibits cyanide-binding behaviour that is similar to that of the wild type, both in its oxidized and reduced forms. However, the rate constant for carbon monoxide recombination increases substantially, suggesting that the access route for carbon monoxide, but not for cyanide, is perturbed by this amino acid substitution.

SUBMITTER: Meunier B

PROVIDER: S-EPMC1219140 | biostudies-other | 1998 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe(41) has been substituted by Trp, Val or Ala and the properties of the mutant proteins have been compared with that of the wild-type. The ferric and ferrous states have been studied by resonance Raman, electronic absorption and Fourier-transform infrared spectroscopies, together with their respective fluoride and CO complexes as probes for the integrity of the distal haem-pocket hydrogen-bonding network. The catalytic properties of the mutants, most notably the HRPC-mutant Phe(41)-->Trp (F41W) variant, were also affected. Structural modelling suggests that the bulky indole group of the F41W mutant blocks the distal cavity, inhibiting the binding of fluoride and CO to the haem iron, severely impairing the reaction of the enzyme with H(2)O(2) to form Compound I. Substitution with the smaller side-chain residues Val or Ala resulted in a 2-fold increase in the affinity of the mutants for the aromatic donor benzhydroxamic acid (BHA) compared with the wild-type, whereas the sterically hindered F41W mutant was not able to bind BHA at all. All the mutations studied increased the amount of a ferric six-coordinate aquo-high-spin species. On the other hand, the similarity in the Fe-Im stretching frequencies of the mutants and wild-type protein suggests that the distal haem-pocket mutations do not cause any substantive changes on the proximal side of the haem. Spectra of the HRPC mutant Phe(41)-->Ala-CO and the HRPC mutant Phe(41)-->Val-CO complexes strongly suggested a weakening of the interaction between CO and Arg(38) due to a secondary rearrangement of the haem relative to helix B. The effects observed for these HRP mutants were somewhat different from those noted recently for the analogous Coprinus cinereus peroxidase (CIP) mutants, particularly the Trp mutant. These differences can be reconciled in part as being due to the smaller size of the distal cavity of HRP compared with that of CIP.

Project description:Cytochrome-c-peroxidase (CCP) contains a five-coordinate heme active site. The reduction potential for the ferric to ferrous couple in CCP is anomalously low and pH dependent (Eo = ~-180 mV vs. S.H.E. at pH 7). The contribution of the protein environment to the tuning of the redox potential of this couple is evaluated using site directed mutants of several amino acid residues in the environment of the heme. These include proximal pocket mutation to residues Asp-235, Trp-191, Phe-202 and His-175, distal pocket mutation to residues Trp-51, His-52, and Arg-48; and a heme edge mutation to Ala-147. Where unknown, the structural changes resulting from the amino acid substitution have been studied by X-ray crystallography. In most cases, ostensibly polar or charged residues are replaced by large hydrophobic groups or alternatively by Ala or Gly. These latter have been shown to generate large, solvent filled cavities. Reduction potentials are measured as a function of pH by spectroelectrochemistry. Starting with the X-ray derived structures of CCP and the mutants, or with predicted structures generated by Molecular Dynamics (MD), predictions of redox potential changes are modeled using the Protein Dipoles Langevin Dipoles (PDLD) method. These calculations serve to model an electrostatic assessment of the redox potential change with simplified assumptions about heme iron chemistry, with the balance of the experimentally observed shifts in redox potential being thence attributed to changes in the ligand set and heme coordination chemistry, and/or other changes in the structure not directly evident in the X-ray structures (e.g. ionization states, specific roles played by solvent species, or conformationally flexible portions of the protein). Agreement between theory and experiment is good for all mutant proteins with the exception of the mutation Arg 48 to Ala, and His 52 to Ala. In the former case, the influence of phosphate buffer is adduced to account for the discrepancy, and measurements made in a bis-tris propane/2-(N-morpholino)ethanesulfonic acid buffer system agree well with theory. For the latter case, an unknown structural element relevant to His-52, and/or solvent influence in the mutant akin to anion binding in the distal pocket (though lacking proof that it is) manifests in this mutant. The use of exogenous (sixth) ligands in dissecting the contributions to control of redox potential are also explored as a pathway for model building.

Project description:BACKGROUND: Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. RESULTS: In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. CONCLUSIONS: This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

Dataset Information

Redox- and anion-linked protonation sites in horseradish peroxidase: analysis of distal haem pocket mutants.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure