Spectral hole burning and selection of conformational substates in chromoproteins.

We investigated spectral holes burnt at 1.5 K into the origins of several tautomeric forms of mesoporphyrin IX-substituted horseradish peroxidase at pH 8 under pressures up to 2 MPa. From the pressure-induced lineshift the compressibility of the apoprotein could be determined. We found that the compressibility changed significantly when measured at different tautomer origins. It was concluded that there must be a correlation between the tautomer configurations of the chromophore and the actual structures of the apoprotein. As a consequence, specific conformational substates of the protein can be selected by optical selection of the associated tautomers.

Spectral hole burning study of protoporphyrin IX substituted myoglobin.

Protoporphyrin IX substituted myoglobin reveals excellent hole burning properties. We investigated the frequency shift of persistent spectral holes under isotropic pressure conditions in a range from 0 to 2.4 MPa. In this range, the protein behaves like an elastic solid. The shift of the holes under pressure shows a remarkable frequency dependence from which the compressibility of the protein can be determined. The compressibility, in turn, allows for an estimation of the equilibrium volume fluctuations. Within the frame of the model used to interpret the pressure data, it is possible to determine the absorption frequency of the isolated chromophore and the associated solvent shift in the protein environment.

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

Mesoporphyrin IX substituted horseradish peroxidase was studied by fluorescence line narrowing and hole burning techniques at cryogenic temperatures. The spectral data reveal that four pyrrole tautomeric configurations of the chromophore are populated within the protein under the influence of irradiation and/or thermal treatment, and the existence of a fifth and a sixth tautomeric configuration is also likely. The relative population of the tautomers changes upon deuterium substitution through modification of the phototransition rate, and also depends on pH, which changes the protonation of neighboring amino acids in the heme pocket. The energy separation of the origins of the tautomers is approximately 100 cm-1. The distribution of barrier heights separating the different tautomeric forms in the ground state and their distribution was determined by temperature cycling hole burning. The distributions can be approximated by Gaussians. The experiment directly yields the distributions on a relative temperature scale, and a model is presented to transform the barrier heights into energy values. It is suggested that the energies for the tautomers are split partially due to the protein crystal field and that the trapping of the tautomeric forms is the consequence of interactions with neighboring amino acids within the heme pocket.

Softening of the packing density of horseradish peroxidase by a H-donor bound near the heme pocket.

We use pressure tuning of spectral holes to estimate the compressibility of protein molecules by optical means. We found that the compressibility of mesoporphyrin-substituted horseradish peroxidase increases by a factor of three when it incorporates small aromatic H-donor molecules that bind in the vicinity of its heme pocket. Such a dramatic softening of its packing density corresponds to a jump from a compressibility range characteristic for the solid state into that characteristic for liquids.

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

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.