Near-UV Circular Dichroism and UV Resonance Raman Spectra of Individual Tryptophan Residues in Human Hemoglobin and Their Changes upon the Quaternary Structure Transition

Abstract

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αβ dimer: at α14, β15, and β37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (β15Trp→Leu), and rHb (β37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and β15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas β37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of β37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.

Probing Tertiary Structure of Proteins Using Single Trp Mutations with Circular Dichroism at Low Temperature

“…24,53 Thus, deconvolution of the CD spectra is a valuable tool to correctly determine intensities, signs, as well as positions of the 1 L a and 1 L b bands of Trp. As recommended previously, 1 inspection of absorption spectra greatly facilitates identification of CD bands.…”

State-by-State Investigation of Destructive Interference in Resonance Raman Spectra of Neutral Tyrosine and the Tyrosinate Anion with the Simplified Sum-over-States Approach

“…[4][5][6][7] RRS has also been used to investigate the interactions between metal atoms in metallo-enzymes [8][9][10] and nearby amino acid residues serving as coordinating ligands. [10][11][12] Moreover, RRS has been used for examining tertiary protein structure such as the α and ψ angles of the peptide linkages in proteins, 13 as well as the hydrophobicity around aromatic amino acid residues. 14 Clearly, the RRS signal is sensitive to intermolecular interactions or any process that affects the electronic structure.…”

Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or β Subunits: Circular Dichroism, 1H NMR, and Resonance Raman Studies

“…We have studied the circular dichroism (CD) spectra of native human adult hemoglobin (Hb A) to elucidate the relationship between its structure and oxygen (O 2 ) binding function . O 2 binding sites of hemoglobin (Hb) and myoglobin (Mb) are heme (Fe‐protoporphyrin IX complex).…”