Protein dynamics in an intermediate state of myoglobin: Optical absorption, resonance Raman spectroscopy, and x-ray structure analysis

A metastable state of myoglobin is produced by reduction of metmyoglobin at low temperatures. This is done either by irradiation with x-rays at 80 K o r by electron transfer from photoexcited tris(2,2'-bipyridine)-ruthenium(II ) at 20 K. At temperatures above 150 K, the conformational transition towar d the equilibrium deoxymyoglobin is observed. X-ray crystallography, Raman spectroscopy, and temperature-dependent optical absorption spectroscopy sho w that the metastable state has a six-ligated iron low-spin center. The x-r ay structure at 115K proves the similarity of the metastable state with met myoglobin. The Raman spectra yield the high-frequency vibronic modes and gi ve additional information about the distortion of the heme. Analysis of the temperature dependence of the line shape of the Soret band reveals that a relaxation within the metastable state starts at similar to 120 K. Paramete rs representative of static properties of the intermediate state are close to those of CO-ligated myoglobin, while parameters representative of dynami cs are close to deoxymyoglobin. Thus within the metastable state the relaxa tion to the equilibrium is initiated by changes in the dynamic properties o f the active site.