title: UV resonance Raman and excited-state relaxation rate studies of hemoglobin.
abstract: We have measured the UV resonance Raman (UVRR) spectra of human methemoglobin fluoride (metHbF) and examined the Raman saturation behavior of the metHbF trytophyl (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 that 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.
authors.affiliation: Department of Chemistry, University of Pittsburgh, Pennsylvania 15260.
authors.forename: N
authors.initials: N
authors.lastname: Cho
authors.forename: S
authors.initials: S
authors.lastname: Song
authors.forename: S A
authors.initials: SA
authors.lastname: Asher
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D006860:Hydrogen Bonding; D008706:Methemoglobin / Q000031:analogs & derivatives* / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: C041160:methemoglobin fluoride; D008706:Methemoglobin