The spectral properties of the protochlorophyllide forms in the epicotyls of dark-grown pea seedlings have been studied in a temperature range, from 10 to 293 K with conventional fluorescence emission and excitation spectroscopy as well as by fluorescence line narrowing (FLN) at cryogenic temperatures. The conventional fluorescence techniques at lower temperatures revealed separate bands at 628, 634-636, 644 and 655 nm. At room temperature (293 K) the 628 and 634-636 nm emission bands strongly overlapped and the band shape was almost independent of the excitation wavelength. Under FLN conditions, vibronically resolved fluorescence spectra could be measured for the 628 and 634-636 nm bands. The high resolution of this technique excluded the excitonic nature of respective excited states and made it possible to determine the pure electronic (0,0) range of the spectra of the two components. Thus it was concluded that the 628 and 634-636 nm (0,0) emission bands originate from two monomeric forms of protochlorophyllide and the spectral difference is interpreted as a consequence of environmental effects of the surrounding matrix. On the basis of earlier results and the data presented here, a model is discussed in which the 636 nm form is considered as an enzyme-bound protochlorophyllide and the 628 nm form as a protochlorophyllide pool from which the substrate is replaced when the epicotyl is illuminated with continuous light.
The heme of horseradish peroxidase is buried in the protein, but a channel from the protein surface connects
the aqueous solution to the heme site. Ferric horseradish peroxidase has an absorption band at 640 nm that
is attributed to a charge-transfer (CT) transition between the a2u HOMO of π electrons of the porphyrin ring
and the d
xy
/d
yz
orbital of the ferric ion. Because the water channel extends to the Fe, it seems likely that the
CT band will be sensitive to the hydration of the protein. To study this premise, the protein was incorporated
into trehalose/sucrose glasses and the hydration of the sugar glasses was varied. Absorption spectra of HRP
in sugar glasses and in glycerol/water were taken in the range 10−300 K. The CT absorption band shows
vibronic fine structure. The peak positions are the same in hydrated sugar and glycerol/water but the peak
positions change in desiccated sugar glass. The data suggest that in hydrated, but not desiccated, sugar glass,
water is retained in the heme pocket. Binding of the competitive inhibitor benzohydroxamic acid to the protein
increases the CT absorption and resolution. The effect of benzohydroxamic acid on the Fe as calculated
using a combination of density functional theory and molecular mechanics is to stabilize the spin state 3/2
with respect to 5/2. At low temperature the widths of the lines in the CT band are narrower for the protein in
glycerol/water (glass transition at ∼150 K) than in trehalose/sucrose (glass formation at 65 °C). This indicates
that the CT band is inhomogeneously broadened and sensitive to the solvent. The spectral narrowing of the
CT absorption occurs as the temperature decreases over the temperature range studied. Water, as indicated
by the OH stretch, also shifts in this range. The findings are discussed in terms of how buried water and
nearby charges can modulate the activity of the heme.
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