Tyrosine and Tryptophan Structure Markers in Hemoglobin Ultraviolet Resonance Raman Spectra: Mode Assignments via Subunit-Specific Isotope Labeling of Recombinant Protein

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Abstract
Phenyl-deuterated tyrosine (Tyr-d4) and indole-deuterated tryptophan (Trp-d5) have been selectively incorporated into hemoglobin (Hb) by expressing the gene in auxotrophic strains of Escherichia coli. Ultraviolet resonance Raman (UVRR) spectra, using 229-nm excitation, show that difference features characteristic of the Hb quaternary R → T transition are not perturbed by the incorporation of the isotopes. All the UVRR bands between 800 and 1700 cm-1 are assigned to either Tyr or Trp except for the 1511 cm-1 band, which had been thought to arise from the Trp 2 × W18 overtone. This band does not shift upon Trp or Tyr labeling but does shift 5 cm-1 in D2O, suggesting assignment to a histidine (His) residue. Its intensification in the T-state is consistent with His protonation. The α- and β-subunits were selectively labeled, by reconstitution of labeled subunits with unlabeled subunits, to make isotope hybrids. Selective Tyr labeling identified the α subunits as the locus of the Y8a upshift observed in Hb, supporting the previous inference that this shift is associated with the T-state H-bond involving the interfacial Tyr α42 [Rodgers, Su, Subramaniam, & Spiro (1992) J. Am. Chem. Soc. 114, 3697]. Selective Trp labeling showed the Trp α14 contributions to the T − R difference spectrum to be negligible and confirmed Trp β37 as the locus of the W3 difference signal, and probably of the remaining Trp signals as well. The observed downshift of W17 and upshift of Wd5 in the T-state are consistent with a stronger T-state H-bond between Trp β37 and Asp α94; the resulting excitation profile red shift accounts for the dominance of the Trp β37 contribution to the T − R difference UVRR spectrum.