J. Biol. Chem., Vol. 261, Issue 4, 1720-1723, Feb, 1986
Heme-linked ionizations in horseradish peroxidase detected by Raman difference spectroscopy
JA Shelnutt, RG Alden and MR Ondrias
Heme-linked ionizations of the acidic and basic isoenzymes of ferrous
horseradish peroxidase influence both the Fe-histidine stretching mode and
the oxidation-state marker line. First, Raman difference spectroscopy of
horseradish peroxidase confirms earlier work showing that v(Fe-His)
undergoes a transition in frequency with a pK that is characteristic of the
enzyme's functional properties. The Fe-histidine mode shifts by about
2.5-3.0 cm-1 for horseradish peroxidase C and by about 6 cm-1 for the
acidic isoenzyme. Further, we find that the oxidation-state marker line v4
also exhibits a transition with the same pK. For horseradish peroxidase C
the shift in v4 is 0.4 cm-1 and the pK is 7.1 +/- .5, in good agreement
with the pK found by other techniques. Shifts in these two Raman lines are
correlated for the pK 7.1 transition and attain their highest frequency at
low pH. The correlation is in marked contrast with R/T shifts in
hemoglobins for which delta v(Fe-His) and delta v4 are also linearly
related but shift in opposite directions. The shift in v4 suggests a
mechanism for pH control of catalytic function based on ring pi-charge
density effects on the energy of charge-depleted high oxidation-state
intermediates. A second transition in v4 (delta v4 = 2.6 cm-1) with a pK of
10.0 is interpreted in terms of a change in ligation and spin state.
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