Photophysics and photochemistry of horseradish peroxidase A2 upon ultraviolet illumination.

Detailed analysis of the effects of ultraviolet (UV) and blue light illumination of horseradish peroxidase A2, a heme-containing enzyme that reduces H(2)O(2) to oxidize organic and inorganic compounds, is presented. The effects of increasing illumination time on the protein's enzymatic activity, Reinheitzahl value, fluorescence emission, fluorescence lifetime distribution, fluorescence mean lifetime, and heme absorption are reported. UV illumination leads to an exponential decay of the enzyme activity followed by changes in heme group absorption.

Crystal structures of the free and sterol-bound forms of beta-cinnamomin.

The crystal structure of the elicitin beta-cinnamomin (beta-CIN) was determined in complex with ergosterol at 1.1 A resolution. beta-CIN/ergosterol complex crystallized in the monoclinic space group P2(1), with unit cell parameters of a = 31.

Formation of a misfolded conformation during refolding of HRPA1 in the presence of calcium.

Horseradish peroxidase A1 can refold to a native-like structure without binding calcium, originating a Ca2+-depleted native state as previously demonstrated. Thermal unfolding studies of horseradish peroxidase anionic 1 (HRPA1) have shown that calcium ions present during refolding lead to the appearance of a misfolded conformational state, which cannot incorporate the heme group. This calcium-induced conformational state, ICa2+, is less stable than the native state and has distinct secondary and tertiary structures as probed by far-UV and visible circular dichroism and tryptophan fluorescence.

Conformational states of HRPA1 induced by thermal unfolding: effect of low molecular weight solutes.

Fluorescence, CD, and activity measurements were used to characterize the different conformational states of horseradish peroxidase A1 induced by thermal unfolding. Picosecond time-resolved fluorescence studies showed a three-exponential decay dominated by a picosecond lifetime component resulting from energy transfer from tryptophan to heme. Upon thermal unfolding a decrease in the preexponential factor of the picosecond lifetime and an increase in the quantum yield were observed approaching the characteristics observed for apoHRPA1.

Heme and pH-dependent stability of an anionic horseradish peroxidase.

Horseradish peroxidase A1 thermal stability was studied by steady-state fluorescence, circular dichroism and differential scanning calorimetry at pH values of 4, 7 and 10. Changes in the intrinsic protein probes, tryptophan fluorescence, secondary structure, and heme group environment are not coincident. The T(m) values measured from the visible CD data are higher than those measured from Trp fluorescence and far-UV CD data at all pH values showing that the heme cavity is the last structural region to suffer significant conformational changes during thermal denaturation.