Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. Mild hypertensive events have been reported during in vivo trials of extracellular protein-based blood substitutes and have been attributed to consumption of NO by hemoglobin and subsequent vasoconstriction due to lack of activation of guanylate cyclase. Thus, understanding the reactions of NO with hemoglobin is crucial to the development of a risk-free extracellular oxygen carrier.
The reactions of NO with recombinant oxy-, deoxy-, and metmyoglobin have been examined in order to determine the chemical mechanisms involved in these processes. Sperm whale myoglobin was chosen as a simple model system for determining the structural and chemical factors that regulate NO reactivity. These myoglobin studies have led to four major conclusions. (1) NO-induced oxidation of MbO$\sb2$ takes place by direct reaction between NO and bound O$\sb2$, and the rate-limiting step is NO diffusion into the distal pocket. (2) In wild-type ferrous myoglobin, the strength of hydrogen bonding between His64(E7) and bound NO is several-fold stronger than hydrogen bonding to bound CO but is $\sim$100-fold weaker than hydrogen bonding to bound O$\sb2$. (3) The pathway for NO entry into the distal pocket of either deoxy- or metmyoglobin appears to involve a channel between Phe43(CD1), the heme-7-propionate, and Thr67(E10) which is opened by outward rotation of the His64(E7) side chain. (4) The rate-limiting step for aerobic oxidation of MbNO is dissociation of NO followed by NO-induced oxidation of newly-formed MbO$\sb2$.
These results have already proved to be applicable to other heme proteins and have led to the design of recombinant hemoglobins which can function as extracellular oxygen-carriers with minimal interference with NO messenger pathways.