Reactivity of Cyanate with Valine-1 (ar) of Hemoglobin

The 3-fold increase in the carbamylation rate of Val-1 (cu) of hemoglobin upon deoxygenation described earlier is now shown to be a sensitive probe of conformational change. Thus, whereas this residue in methemoglobin A is carbamylated at the same rate as in liganded hemoglobin, upon addition of inositol hexaphosphate its carbamylation rate is enhanced 30% as much as the total change in the rate between the CO and deoxy states. For CO-hemoglobin Kansas in the presence of the organic phosphate, the relative increase in the carbamylation rate of this residue is about 50%. These results indicate that methemoglobin A and hemoglobin Kansas in the presence of inositol hexaphosphate do not assume a conformation identical with deoxyhemoglobin but rather form either a mixture of R and T states or an intermediate conformation in the region around Val-1 (cu). Studies on the mechanism for the rate enhancement in deoxyhemoglobin suggest that the cyanate anion binds to groups in the vicinity of Val-1 (LY) prior to proton transfer and carbamylation of this NH,-terminal residue. Thus, specific removal with carboxypeptidase B of Arg-141 c(u), which is close to Val-1 c(u) in deoxyhemoglobin, abolishes the enhancement in carbamylation. Chloride, which has the same valency as cyanate, is a better competitive inhibitor of the carbamylation of deoxyhemoglobin (K, = 50 mu) compared with liganded hemoglobin. Nitrate and iodide are also effective inhibitors of the carbamylation of Val-1 ((u) of deoxyhemoglobin (Ki = 35 mu); inorganic phosphate, sulfate, and fluoride are poor competitive inhibitors. The change in pK, of Val-1 c(u) upon deoxygenation may be due to its differential interaction with chloride.