Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase

Spectroscopy of horseradish peroxidase with and without the substrate analo g, benzohydroxamic acid, was monitored in a glycerol/water solvent as a fun ction of temperature. It was determined from the water infrared (IR) absorp tion that the solvent has a glass transition at 170-180 K. In the absence o f substrate, both the heme optical Q(0,0) absorption band and the IR absorp tion band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, wherea s the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate st rongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution t o the Q(0,0) bandwidth arises from the heme vibrations that are not affecte d by the phase transition. The CO band thermal broadening stems from the an harmonic coupling with motions of the heme environment, which, in the glass y state, are frozen in. Unusually strong temperature broadening of the CO b and is interpreted to be caused by thermal population of a very flexible ex cited conformational substrate. Analysis of literature data on the thermal broadening of the A, band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26 :337-355) shows that such a state presents itself also in myoglobin.