TYROSINE AND TRYPTOPHAN STRUCTURE MARKERS IN HEMOGLOBIN ULTRAVIOLET RESONANCE RAMAN-SPECTRA - MODE ASSIGNMENTS VIA SUBUNIT-SPECIFIC ISOTOPELABELING OF RECOMBINANT PROTEIN

Phenyl-deuterated tyrosine (Tyr-d(4)) and indole-deuterated tryptophan (Trp-d(5)) have been selectively incorporated into hemoglobin (Hb) by expressing the gene in auxotrophic strains of Escherichia coil. Ultra violet resonance Raman (UVRR) spectra, using 229-nm excitation, show t hat difference features characteristic of the Hb quaternary R-->T tran sition 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 f rom the Trp 2 x W18 overtone. This band does not shift upon Trp or Tyr labeling but does shift 5 cm(-1) in D2O, suggesting assignment to a h istidine (His) residue. Its intensification in the T-state is consiste nt with His protonation. The alpha- and beta-subunits were selectively labeled, by reconstitution of labeled subunits with unlabeled subunit s, to make isotope hybrids. Selective Tyr labeling identified the ct s ubunits as the locus of the Y8a upshift observed in Hb, supporting the previous inference that this shift is associated with the T-state H-b ond involving the interfacial Tyr alpha 42 [Rodgers, Su, Subramaniam, & Spiro (1992) J. Am. Chem. Sec. 114, 3697]. Selective Trp labeling sh owed the Trp alpha 14 contributions to the T-R difference spectrum to be negligible and confirmed Trp beta 37 as the locus of the W3 differe nce signal, and probably of the remaining Trp signals as well. The obs erved downshift of W17 and upshift of Wd5 in the T-state are consisten t with a stronger T-state H-bond between Trp beta 37 and Asp alpha 94; the resulting excitation profile red shift accounts for the dominance of the Trp beta 37 contribution to the T-R difference UVRR spectrum.