The reaction of oxyhemoglobin with nitric oxide: EPR evidence for an iron(III)-nitrate intermediate

The reaction of oxyhemoglobin with nitric oxide is known to occur in vivo, with direct medical and pathophysiological relevance. The mechanism has been proposed to involve a transient iron(III)-peroxynitrite intermediate, the formation of which would be rate-limiting and the decay of which would yield iron(III)-aqua/iron(III)-hydroxo hemoglobin. Reported here are rapid-freeze-quench EPR (RFQ-EPR) spectroscopy data on the reaction of oxyhemoglobin with nitric oxide; no direct evidence is seen for an iron(III)-peroxynitrite intermediate. These findings are consistent with theoretical considerations according to which such an intermediate does form but is too short-lived to be detectable. Instead, iron(III) low-spin and high-spin transient species are detected in the RFQ-EPR experiments, which can be explained as arising from interactions of nitrate with methemoglobin.