Three mutants of horseradish peroxidase isoenzyme C (HRPC) have been constructed in which the conserved distal aromatic residue Phe41 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 Phe41→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 H2O2 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 Phe41→Ala—CO and the HRPC mutant Phe41→Val—CO complexes strongly suggested a weakening of the interaction between CO and Arg38 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.
- distal mutants
- haem protein
- horseradish peroxidase
- resonance Raman spectroscopy
Abbreviations used: ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate); BHA, benzhydroxamic acid; 5c, five coordinate; 6c, six coordinate; CCP, cytochrome c peroxidase; CIP, Coprinus cinereus peroxidase, expressed in Aspergillus oryzae (identical with Arthromyces ramosus peroxidase); CT1 band, long-wavelength (>600nm) porphyrin-to-metal charge-transfer band; HRPC, horseradish peroxidase isoenzyme C; F41A, HRPC mutant Phe41→Ala; F41V, HRPC-mutant Phe41→Val; F41W, HRPC-mutant Phe41→Trp; F54G, CIP-mutant Phe54→Gly; F54V, CIP-mutant Phe54→Val; F54W, CIP-mutant Phe54→Trp; FTIR, Fourier-transform infrared; Vis, visible; HRPA1, horseradish peroxidase isoenzyme A1; HS, high spin; Mops, 3-(N-morpholino)propanesulphonic acid; QS, quantum mechanically mixed spin; RR, resonance Raman.
- The Biochemical Society, London ©2002