Recombinant horseradish peroxidase isoenzyme C: the effect of distal haem cavity mutations (His42→Leu and Arg38→Leu) on compound I formation and substrate binding

1996 ◽  
Vol 1 (2) ◽  
pp. 136-142 ◽  
Author(s):  
Jose Neptuno Rodriguez-Lopez ◽  
Andrew T. Smith ◽  
R. N. F. Thorneley
Keyword(s):  
Horseradish Peroxidase ◽  
Substrate Binding ◽  
Compound I ◽  
Peroxidase Isoenzyme ◽  
Recombinant Horseradish Peroxidase

Spectroscopic characterization of mutations at the Phe41 position in the distal haem pocket of horseradish peroxidase C: structural and functional consequences

2002 ◽  
Vol 363 (3) ◽  
pp. 571-579 ◽  
Author(s):  
Hendrik A. HEERING ◽  
Andrew T. SMITH ◽  
Giulietta SMULEVICH
Keyword(s):  
Horseradish Peroxidase ◽  
Fold Increase ◽  
Spectroscopic Characterization ◽  
Wild Type ◽  
Compound I ◽  
Peroxidase Isoenzyme ◽  
Mutant Proteins ◽  
Aromatic Donor ◽  
Form Compound ◽  
Trp Mutant

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.

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

1986 ◽  
Vol 64 (4) ◽  
pp. 323-327 ◽  
Author(s):  
Mohammed A. Kashem ◽  
H. Brian Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Ph Dependence ◽  
Transient State ◽  
Nicotinamide Adenine ◽  
Compound I ◽  
Single Ionization ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Kinetics Of

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 °C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I–NADH reaction can be explained in terms of a single ionization of pKa = 4.7 ± 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-H–NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 ± 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II–NADH reaction was observed. Over the pH range of 4–10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 × 105 to5.6 × 102 M−1 s−1 and of HRP-II with NADH varied from 4.4 × 104 to 4.1 M−1 s−1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.

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