Time-resolved and static resonance Raman spectroscopy of horseradish peroxidase intermediates.
By using pulsed and continuous wave laser irradiation in the 350-450-nm region, we have characterized Raman scattering from horseradish peroxidase (HRP) compounds I and II and from iron porphyrin pi-cation radical model compounds. For compound II we support the suggestion [Terner, J., Sitter, A. J., & Reczek, C. M. (1985) Biochim. Biophys. Acta 828, 73-80; Proniewicz, L. M., Bajdor, K., & Nakamoto, K. (1986) J. Phys. Chem. 90, 1760-1766] that resonance enhancement of the FeIV = O vibration proceeds by way of a charge-transfer state. Our excitation profile data locate this state at approximately 400 nm. Compound I was prepared at neutral pH by rapid mixing of the resting enzyme with hydrogen peroxide. Each sample aliquot was excited by a single, 10-ns laser pulse to generate the Raman spectrum; optical spectroscopy following the Raman measurement confirmed that HRP-I was the principal product during the time scale of the measurement. The Raman spectrum of this species, however, is not characteristic of that which we observe from metalloporphyrin pi-cation radicals [Oertling, W. A., Salehi, A., Chung, Y., Leroi, G. E., Chang, C. K., & Babcock, G. T. (1987) J. Phys. Chem. 91, 5887-5898], including the iron porphyrin cation radicals reported here. Instead, the spectrum recorded for HRP-I at neutral pH is suggestive of an oxoferryl heme with the same geometric and electronic structure as that of HRP-II at high pH.(ABSTRACT TRUNCATED AT 250 WORDS)
Oertling
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Department of Chemistry, Michigan State University, East Lansing 48824-1322.
Babcock
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D006735:Horseradish Peroxidase / Q000378:metabolism*; D007700:Kinetics; D007834:Lasers; D008962:Models, Theoretical; D010544:Peroxidases / Q000378:metabolism*; D011166:Porphyrins / Q000032:analysis; D011487:Protein Conformation; D013059:Spectrum Analysis, Raman / Q000379:methods; D013997:Time Factors
D011166:Porphyrins; D006735:Horseradish Peroxidase; D010544:Peroxidases