Kinetic Analysis and Structural Interpretation of Competitive Ligand Binding for NO Dioxygenation in Truncated Hemoglobin†N.

ABSTRACT

The conversion of nitric oxide (NO) into nitrate (NO3- ) by dioxygenation protects cells from lethal NO. Starting from NO-bound heme, the first step in converting NO into benign NO3- is the ligand exchange reaction FeNO+O2 →FeO2 +NO, which is still poorly understood at a molecular level. For wild-type (WT) truncated hemoglobin N (trHbN) and its Y33A mutant, the calculated barriers for the exchange reaction differ by 1.5 kcal mol-1 , compared with 1.7 kcal mol-1 from experiment. It is directly confirmed that the ligand exchange reaction is rate-limiting in trHbN and that entropic contributions account for 75 % of the difference between the WT and the mutant. Residues Tyr 33, Phe 46, Val 80, His 81, and Gln 82 surrounding the active site are expected to control the reaction path. By comparison with electronic structure calculations, the transition state separating the two ligand-bound states was assigned to a 2 A state.