PH-INDUCED CONFORMATIONAL PERTURBATION IN HORSERADISH-PEROXIDASE - PICOSECOND TRYPTOPHAN FLUORESCENCE STUDIES ON NATIVE AND CYANIDE-MODIFIED ENZYMES

The fluorescence-decay characteristics of the single tryptophan present in horseradish peroxidase (HRP) have been studied using dye-laser pulses and single-photon counting techniques. The decay was found to be dominated by a picosecond-lifetime component, with small contributions from two other lifetime components in the nanosecond range. The distance of the tryptophan residue was estimated from the fluorescence-energy transfer to the heme moiety using Forster's theory. The tryptophan residue was found to be approximately 1.2 nm from the heme moiety at neutral pH. Detailed analysis of the fluorescence-decay profiles using the maximum-entropy method (MEM) has been carried out. The results of the MEM analysis also showed a maximum amplitude peak at approximately 45 ps (at pH approximate to 7) with a very small (< 5%) contribution from two other components. Similar results were obtained with the cyanide derivative of the enzyme (HRPCN) where the major lifetime component was found to be 58 ps at neutral pH. The picosecond component of fluorescence lifetimes of native HRP as well as of HRPCN were found to increase with decrease in pH in the range pH 6-3.5. Moreover, the native enzyme showed significant increase in the magnitude of this fast lifetime component at pH above 8. Such increase in the major lifetime component possibly indicated a conformational perturbation caused by pH change in the enzyme. However, the pH dependence of HRPCN, which is devoid of alkaline transition, showed that the shortest lifetime component remains almost unchanged over the pH range 6-11. This result showed that the alkaline transition in native HRP is associated with a structural change in the distal region of the heme center, which is absent in the cyanide-ligated enzyme. The results have been discussed with respect to understanding the pH-induced effects associated with salt bridge and hydrogen-bonding network in HRP.