Resonance Raman spectroscopic evidence for heme iron-hydroxide ligation in peroxidase alkaline forms.
Horseradish peroxidase will convert from a five-coordinate high-spin heme at neutral pH to a six-coordinate low-spin heme at alkaline pH. Though alkaline forms of other heme proteins such as hemoglobin and myoglobin are known to contain a heme-ligated hydroxide, alkaline horseradish peroxidase has been considered not to contain a ligated hydroxide. Several alternatives have been proposed which would be stronger field ligands than a hydroxide ion. In this report we provide resonance Raman evidence, using Soret excitation, that alkaline horseradish peroxidase does in fact contain a heme iron-ligated hydroxyl group. The band was located for isoenzymes C and A-1 by its sensitivity to 18O substitution and confirmed with 54Fe, 57Fe, and 2H. An isoenzyme of turnip peroxidase was investigated and found to also contain a ligated hydroxide at alkaline pH. The observed peroxidase Fe(III)-OH frequencies are 15-25 cm-1 higher than the corresponding frequencies of alkaline methemoglobin and metmyoglobin and correlate with changes in spin-state distribution. This is explained in the context of hydrogen bonding to a distal histidine which results in increased ligand field strength facilitating the formation of low-spin hemes. It has been demonstrated that the ferryl/ferric redox potential of horseradish peroxidase is markedly lowered at alkaline pH (Hayashi, Y., and Yamazaki, I. (1979) J. Biol. Chem. 254, 9101-9106). These observations are rationalized in terms of oxidation of a ligated ferric hydroxyl group facilitated through base catalysis by a distal histidine.
Department of Chemistry, Virginia Commonwealth University, Richmond 23284-2006.
A J
AJ
Sitter
J R
JR
Shifflett
J
J
Terner
D006418:Heme / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000378:metabolism*; D007501:Iron / Q000378:metabolism*; D010544:Peroxidases / Q000378:metabolism*; D013059:Spectrum Analysis, Raman
D006878:Hydroxides; D006418:Heme; D007501:Iron; D006735:Horseradish Peroxidase; D010544:Peroxidases