UV resonance Raman spectra reveal a structural basis for diminished proton and CO2 binding to alpha,alpha-cross-linked hemoglobin.

UV resonance Raman difference spectra between ligated and deoxyhemoglobin contain tryptophan and tyrosine signals which arise from quaternary H-bonds in the T state, which are broken in the R state. These H-bonds are unaffected by bis(3,5-dibromosalicyl) fumarate cross-linking at the Lys alpha 99 residues, which prevents dissociation of Hb tetramers to dimers. However, when the pH is lowered from 9.0, or when NaCl is added, intensity is diminished for the tyrosine Y8 and tryptophan W3 bands of cross-linked deoxyHb, but not of native deoxyHb. This effect is attributed to weakening of tertiary H-bonds involving Tyr alpha 140 and Trp alpha 14, when the T state salt bridge between Val alpha 1 and Arg alpha 141 is formed via protonation of the terminal amino group and anion binding. The Tyr alpha 140-Val alpha 93 H-bond connects the Arg alpha 141-bearing H helix with the Lys alpha 99-bearing G helix. Weakening of the H-bond reflects a tension between the fumarate linker and the salt-bridge. This tension inhibits protonation of the Val alpha 1 amino terminus, thus accounting for the diminution of both proton [Bohr effect] and CO2 binding in the T state as a result of cross-linking.