Heme protein structure and dynamics, studied by Resonance Raman Spectroscopy

Abstract

Resonance Raman spectroscopy provides a structure probe for the iron porphyrin complex of heme proteins. It is capable of monitoring the time evolution of the structure when carried out with pulsed laser excita— tion, and/or flow techniques. The prompt photoproduct of COHb(Hb — hemoglo— bin), produced and detected with 30 Ps pulses from a synchronously pumped mode—locked dye laser showed a RR spectrum with po9hyrin skeletal frequencies characteristic of an in—plane higl'—spin Fe I heme. This result implies that photoexcitation is followed by intersystem crossing to a highspin ligand—field state of COBb, which is dissociative with respect to CO: the Fe atom, however, is constrained from moving out of the heme plane to the normal deoxy—heme structureS The subsequent out-of—plane relaxation was monitored via the porphyrin skeletal frequencies in pulsed laser and flow experiments; its time constant was bracketed between 20 and 300 ns, much longer than expected for simple atomic displacements. Since X—ray crystallography has shown that deligation of Hb is accompanied by movement (lA) of the entire F helix to which the proximal imidazole ligand is attached, it can be inferred that the out—of-plane Fe relaxation requires a large scale protein conformation change, to which it is coupled. This coupled motion may be part of the molecular mechanism for the R -T transition, which occurs later. The O2Hb photointermediate, produced and detected by higher intensity (5 nj 30 ps YAG pulses showed a different RE spectrum, with substantially down—shifted skeletal frequencies, suggestive of an electronically excited state with Tr* orbital occupation. This spectrum relaxes rapidly, to one similar to that of the COBb photoproduct. The iron—imidazole stretching mode has been located in the RR spectrum of R—' state deoxyHb using flow techniques, and found to be symmetrical (nearly equivalent u and chain contributions) and upshif ted relative to T—state deoxyHb, as is observed in chemically modified Hb's. The R—T frequency difference represents an appreciable bond energy change, which may be due to molecular strain or to changes in H—bonding.