Further studies on the kinetics and equilibria of the reactions of nitric oxide with haemoproteins

Reduced haemoglobin is known to react faster with NO than with either O$_{2}$ or CO, and the reaction has now been studied in more detail to allow comparisons of the degree of haem-haem interaction with the different ligands. As a kinetic measure of the interaction, the observed ratio of x$_{4}^{\prime}$ to x$_{1}^{\prime}$ (x$_{1}^{\prime}$, x$_{4}^{\prime}$ being the respective velocity constants of the combination of the first and fourth ligand molecules) is divided by the value of the ratio to be expected on statistical principles, namely 0$\cdot $25. It has been found that the extent of interaction, as determined in this way, is inversely related to the rate of combination of the first ligand molecule, and for CO, O$_{2}$ and NO it is 80, 12 and 3$\cdot $3 respectively. The rate constants for the combination of sheep haemoglobin with the first and fourth molecules of NO are 3 $\times $ 10$^{7}$ and 2$\cdot $5 $\times $ 10$^{7}$ M$^{-1}$ S$^{-1}$. Determinations of the rate of combination with, and dissociation of NO from, horse myoglobin (Mgb) at 22 degrees C give 1$\cdot $7 $\times $ 10$^{7}$ M$^{-1}$ S$^{-1}$ and 7 $\times $ 10$^{-5}$ S$^{-1}$ for the corresponding velocity constants. The expected concentration of free NO for half saturation is thus 4 $\times $ 10$^{-12}$ M corresponding to pNO of 1$\cdot $5 $\times $ 10$^{-6}$ mmHg. The competitive equilibrium of myoglobin between NO and CO has been directly measured and is about 9000 at 19 degrees C (in favour of NO). This value agrees fairly well with that calculated indirectly from the measured values of the velocity constants of the reactions CO + Mgb $\rightleftharpoons $ COMgb and NO + Mgb $\rightleftharpoons $ Mgb.