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-2,2′-bipyridine ruthenium (II) using 1.2-μs laser pulses (λ=460 nm, 33 mJ/pulse). Upon reduction an intermediate state of the protein is formed where the reduced Fe²⁺ atom retains its bond with 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 low temperature (T=90 K) by reduction of metMb using thermalized electrons. Photolysis of the Fe²⁺-H₂O bond of the low-spin protein intermediate state at T=20 K was observed after illumination with a laser diode (λ=685 nm, 24 mW). Upon photolysis, a secondary high-spin intermediate state is produced which is metastable at T≤20 K. Above 20 K, the photodissociated water molecule rebinds with the Fe²⁺ atom, and the protein returns to the low-spin intermediate conformation. For T>100 K, a blue-shift 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. Above 140 K, an irreversible nonexponential (vs. time) relaxation of the low-spin intermediate conformation to deoxymyoglobin is observed.