Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase.
Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.
Nissum
M
M
Department of Chemistry, Odense University, Denmark.
Neri
F
F
Mandelman
D
D
Poulos
T L
TL
Smulevich
G
G
D060387:Ascorbate Peroxidases; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D005290:Ferric Compounds / Q000737:chemistry; D007093:Imidazoles / Q000737:chemistry; D018532:Pisum sativum / Q000201:enzymology*; D010544:Peroxidases / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D005290:Ferric Compounds; D007093:Imidazoles; D010940:Plant Proteins; D011994:Recombinant Proteins; C029899:imidazole; D010544:Peroxidases; D060387:Ascorbate Peroxidases; D003578:Cytochrome-c Peroxidase