Identification of a critical phenylalanine residue in horseradish peroxidase, Phe179, by site-directed mutagenesis and 1H-NMR: implications for complex formation with aromatic donor molecules.
The functional and structural significance of Phe179 of horseradish peroxidase isoenzyme C (HRP C) has been investigated by site-directed mutagenesis. This residue is located in a structurally variable insertion between helices F and G, a motif unique to peroxidases of higher plants. Results obtained for three recombinant enzymes, with Phe179 substituted by Ala, His, or Ser, provide the first demonstration of the importance of this side chain for the binding of aromatic donor molecules. Experimental parameters for direct comparison with the wild-type enzyme were obtained by extensive solution state characterization using both optical and 1H-NMR spectroscopy. Significant chemical shift variations for resonances associated with the exposed heme edge, notably heme methyl C18H3 and heme propionate C17(1)H2, were recorded in NMR spectra of both the resting and cyanide-ligated states of the three Phe179 mutants. Furthermore, comparison of NOE connectivities in NOESY spectra of cyanide-ligated wild-type and mutant enzymes enabled the elusive assignment of the aromatic side chain in close proximity to heme methyl C18H3 to be made to Phe179. Replacement of Phe179 by Ala resulted in an 80-fold decrease in the binding affinity of the cyanide-ligated enzyme for benzhydroxamic acid, with a Kd value similar to that determined for cyanide-ligated HRP A2 (an acidic isoenzyme with valine at position 179). The binding affinity of Phe179-->Ser was similarly decreased, while that of Phe179-->His was partially restored relative to wild-type HRP C. Cyanide-ligated Phe179-->His HRP C exhibited a unique pH-dependent spectral transition associated with a pKa value of 6.5 +/- 0.2, assigned to the His179 side chain. Two closely related enzyme forms exhibiting different affinities for benzhydroxamic acid were observed at neutral pH and above, indicating that the protonation state of His179 gave rise to microheterogeneity in the aromatic donor molecule binding site.
Veitch
N C
NC
Jodrell Laboratory, Royal Botanic Gardens, surrey, U.K. n.veitch@rbgkew.org.uk
Gao
Y
Y
Smith
A T
AT
White
C G
CG
D001565:Benzoates / Q000378:metabolism*; D019817:Benzoic Acid; D003486:Cyanides / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006877:Hydroxamic Acids / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D009626:Terminology as Topic
D001565:Benzoates; D003486:Cyanides; D006877:Hydroxamic Acids; D007527:Isoenzymes; D011522:Protons; D011994:Recombinant Proteins; D006418:Heme; D010649:Phenylalanine; D019817:Benzoic Acid; D006735:Horseradish Peroxidase; C006696:benzohydroxamic acid