Conformational relaxation of a low-temperature protein as probed by photochemical hole burning. Horseradish peroxidase

Abstract

For the first time, conformational relaxation processes have been measured in a small protein, mesoporphyrin-horseradish peroxidase via their influence on spectral diffusion broadening of holes burnt in the fluorescence excitation spectrum of free base mesoporphyrin. Holes were burnt in three 0----0 bands of different tautomeric forms of the chromophore at 1.5 and 4 K, and the spectral diffusion broadening was measured in temperature cycling experiments between 4 and 30 K. The inhomogeneous linewidth for the tautomeric 0----0 bands was estimated to be 60-70 cm-1; the hole width was found narrow, being in the order of 350 MHz (10(-2) cm-1) at 1.5 K what allowed for an extremely sensitive detection of the conformational changes. Though proteins have many features in common with glasses, the spectral diffusion broadening of photochemical holes under temperature cycling conditions in mesoporphyrin horseradish peroxidase has a very different pattern as a function of temperature. Up to 12 K, the linewidth did not significantly change, then around 14 K; a steplike broadening was observed for all three tautomers, although to a different extent. The total magnitude of line broadening up to 30 K was large and also different for the tautomers. We argue that the difference between the behavior of this protein and that of glassy matrices originate from finite size effects; the protein may be characterized by a small number of TLS, and their distribution may bear discrete features.

Allosteric Effectors Influence the Tetramer Stability of Both R- and T-states of Hemoglobin A

“…This change of the electronic transition energy is supposed to be proportional to the solvent shift at atmospheric pressure. If the interaction of the chromophore with the atomic groups of the protein matrix is dominated by one kind of interaction with a potential energy of 1/R n distance dependence, one observes a linear shift of the transition energy as a function of pressure (52)(53)(54). We observed a linear red shift in both cases that has also been reported for other heme proteins (47,49).…”

More than two pyrrole tautomers of mesoporphyrin stabilized by a protein. High resolution optical spectroscopic study

“…We have shown earlier that MP substitution for the heme in HRP does not destroy its substrate binding ability (6). We also demonstrated that at cryogenic temperatures, the inhomogeneous distribution of (0, 0) energy for MP-HRP is narrow, 60 cm-1 (7,8), that is, MP has a well defined location within the protein crevice. The low bimolecular quenching rate of molecular oxygen determined by the fluorescence of protoporphyrin substituted horseradish peroxidase (9) also shows that the heme pocket of this protein has an unusually well protected, rigid structure.…”

Cryoradiolysis and Cryospectroscopy for Studies of Heme-Oxygen Intermediates in Cytochromes P450

“…Absorption spectra and MCD of unstable high-valent intermediates in heme enzymes have been described by Gasyna, Stillman and coworkers (76,89-91). Application of cryogenic optical spectroscopy to biophysical chemistry of proteins, including heme enzymes, has been described by Vanderkooi, Friedrich and colleagues (11,34,92-98). Frauenfelder and colleagues systematically applied various spectroscopic methods at cryogenic temperatures to study fundamental aspects of protein structure and dynamics (12,99-101).…”