UV RESONANCE RAMAN AND EXCITED-STATE RELAXATION RATE STUDIES OF HEMOGLOBIN

We have measured the UV resonance Raman (UVRR) spectra of human methemoglobin fluoride (metHbF) and examined the Raman saturation behavior of the metHbF tryptophyl (Trp) and tyrosyl (Tyr) residues. Our high-quality UVRR spectra devoid of Raman saturation with 229- and 238.3-nm CW laser excitation allow us to determine small changes in Trp and Tyr residue Raman band frequencies and intensities caused by the hemoglobin R-T quaternary structural change induced by the allosteric effector inositol hexaphosphate. At 238.3-nm excitation, we observe a ca. 15 and 8% intensity increase for the Trp and Tyr bands, respectively, upon the R-T transition. In contrast, a small intensity decrease is observed with 225-nm excitation. These intensity alterations result from Trp and Tyr absorption and Raman excitation profile red-shifts which correlate with a strong 231.5-nm R-T absorption spectral change. These absorption and Raman excitation profile red-shifts and our model compound absorption studies together suggest a T-state increase in the hydrogen bond donation of the Trp-beta(2)37 and Tyr-alpha(1)42 residues at the alpha(1) beta(2) subunit interface. The Tyr-alpha 42 residue appears to be a hydrogen bond donor, rather than an acceptor. We determined the electronic excited-state relaxation rates of the Trp and Tyr residues in hemoglobin by using Raman saturation spectroscopy with 225-nm pulsed laser excitation. The observed average excited-state relaxation rate of the Trp residues is ca. 1/120 ps and is independent of the quaternary structure. This rate is slower than that observed for Trp residues of horse myoglobin. The average excited-state relaxation rate of the Tyr residues is ca. 1/60 ps for both the R and T quaternary forms. These are the first Tyr relaxation rates measured for any heme protein.