Class I heme peroxidases: characterization of soybean ascorbate peroxidase.
An efficient expression system [D. A. Dalton et al. Arch. Biochem. Biophys. 328, 1-8, 1996) for soybean nodule ascorbate peroxidase (APX) has, for the first time, been used to generate enzyme in large enough quantities for detailed biophysical analysis. The recombinant APX has been characterized by electronic absorption, EPR, NMR and circular dichroism spectroscopies, and by electrochemistry. Electronic, EPR, and NMR spectra are consistent with a high-spin ferric resting state for the enzyme at 298 K. Low-temperature EPR (7 K) and electronic absorption (77 K) experiments indicate formation of a low-spin heme derivative at these temperatures. The midpoint reduction potential for the Fe(III)/Fe(II) redox couple, determined by spectroelectrochemistry, is -159 +/- 2 mV vs SHE (pH 7.0, 25.0 degrees C, mu = 0.10 M). Circular dichroism spectra of pea and soybean APXs are very similar, indicating common structural features for the two enzymes. The melting temperature of soybean APX, as monitored by circular dichroism spectroscopy, is 49 degrees C. These results represent the first detailed spectroscopic and electrochemical analysis of soybean ascorbate peroxidase and are discussed in the broader context of other class I peroxidases.
Jones
D K
DK
Department of Chemistry, University of Leicester, University Road, Leicester, LE1 7RH, England, United Kingdom.
Dalton
D A
DA
Rosell
F I
FI
Raven
E L
EL
D000595:Amino Acid Sequence; D060387:Ascorbate Peroxidases; D002942:Circular Dichroism; D004563:Electrochemistry; D004795:Enzyme Stability; D005290:Ferric Compounds / Q000378:metabolism; D006418:Heme / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D018532:Pisum sativum; D010544:Peroxidases / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D016415:Sequence Alignment; D013025:Glycine max / Q000201:enzymology*; D013057:Spectrum Analysis; D013696:Temperature
D005290:Ferric Compounds; D006418:Heme; D010544:Peroxidases; D060387:Ascorbate Peroxidases