Insights into Hemoglobin Dynamics from Resonance Raman Spectroscopy

Abstract

Resonance Raman (RR) spectroscopy provides a useful technique for monitoring local structure in complex molecular systems via selective enhancement of the vibrational spectra of chroraophoric centers. The availability of reliable high-power pulsed lasers makes possible two important extensions of the technique: 1) Using nonlinear optical devices, the laser wavelength can be shifted far into the ultraviolet region, allowing simple conjugated chroraophores to be probed, including aromatic side chains of proteins and the peptide bonds of the protein backbone. 2) The temporal characteristics of the pulses can be used to study photochemical transients on the nanosecond, and even the picosecond time scale. These new techniques have been applied to hemoglobin, in order to study the coupling of ligation with protein structure change. Time-resolved studies of the heme RR spectrum establish that photolysis of bound CO leads to very rapid (< 30ps) formation of high-spin heme, via intersystem crossing. This early photoproduct is structurally unrelaxed, however. The Fe-histi-dine bond is compressed, and the excursion of the Fe atom from the heme plane is restricted. The full out-of-plane displacement is associated with a ~ 100ns relaxation, while the Fe-histidine stretching frequency relaxes to the R state deoxyHb value at ~ 1μs, and is further weakened in a 20μs process, which has been assigned to the R→T quaternary rearrangement. When, however, UV excitation, at 218 and 200nm, is used to probe the RR spectra of tryptophan and tyrosine, respectively, evidence is found for the establishment of intersubunit contacts which are characteristic of the T quaternary structure within 7ns of CO photolysis. This result suggests that there is a quite rigid connection between the heme and the interface, leading to very rapid adjustment of the subunit contacts; this must produce a strained structure, which relaxes through subsequent conformational changes to the equilibrium T quaternary structure. A scheme for HbCO photodynamics is presented which incorporates these findings.