Photoinitiated generation of reduced myoglobin intermediates at cryogenic temperatures and their relaxation

Sperm whale myoglobin was reduced within the temperature range of 20-180 K by photoexcitation of tris-a,2'-bipyridine ruthenium (II) using 1.2-mu s la ser pulses (lambda=460 nm, 33 mJ/pulse). Upon reduction an intermediate sta te of the protein is formed where the reduced Fe2+ atom retains its bond wi th the water molecule from the metmyoglobin conformation but is low-spin (S =0), It is shown that the photogenerated reduced intermediate conformation of myoglobin is identical to the low-spin intermediate state produced at lo w temperature (T=90 K) by reduction of metMb using thermalized electrons. P hotolysis of the Fe2+-H2O bond of the low-spin protein intermediate state a t T=20 K was observed after illumination with a laser diode (lambda=685 nm, 24 mW). Upon photolysis, a. secondary high-spin intermediate state is prod uced which is metastable at T less than or equal to 20 K. Above 20 K, the p hotodissociated water molecule rebinds with the Fe2+ atom, and the protein returns to the low-spin intermediate conformation. For T>100 K, a blue-shif t of the Soret band of the low-spin intermediate state is observed that is indicative of structural changes occurring in the vicinity of the heme. Abo ve 140 K, an irreversible nonexponential (vs. time) relaxation of the low-s pin intermediate conformation to deoxymyoglobin is observed.