β93 Modified Hemoglobin:  Kinetic and Conformational Consequences

The reactive sulfhydryl on Cys β93 in human adult hemoglobin (HbA) has been the focus of much attention. It has purported functional roles such as a transporter of nitric oxide and a detoxifier of super oxide. In addition, it has a proposed role in the allosteric mechanism. The present study addresses the functional and conformational consequences of modifying the β93 sulfhydryl using either maleimide or disulfide-based reactions. The geminate and bimolecular recombination of CO derivatives of several different β93-modified Hbs in both solution and sol−gel matrixes provide a window into functional modifications associated with both the R and T states of these proteins. Nanosecond time-resolved visible resonance Raman spectroscopy is used to probe conformational consequences associated with the proximal heme environment. The results show functional and conformational consequences that depend on the specific chemistry used to modify β93. Maleimide-based modification show the most significant alterations of R-state properties including a consistent pattern of a reduced geminate yield and a loss of the favorable heme-proximal histidine interaction normally seen for liganded R-state HbA. A mechanism based on a displacement of the side chain of Tyr β145 is explored as a basis for this effect as well as other situations where there is loss of the quaternary enhancement effect. The quaternary enhancement effect refers to the enhancement of ligand binding properties of the αβ dimers when they are associated into the R-state tetramer.