Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation

Abstract Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant ( k off ). Since no techniques are available to estimate k off , we report herewith a method to determine k off based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The k off value for cyanide dissociation from ferric cyanide horse heart myoglobin (Mb(III)–cyanide) was determined at pH 9.2 and 20.0 °C. Mixing Mb(III)–cyanide and NO solutions brings about absorption spectral changes reflecting the disappearance of Mb(III)–cyanide with the concomitant formation of ferrous nitrosylated Mb. Since kinetics of reductive nitrosylation of Mb(III) is much faster than Mb(III)–cyanide dissociation, the k off value, representing the rate-limiting step, can be directly determined. The k off value obtained experimentally matches very well to that calculated from values of the second-order rate constant ( k on ) and of the dissociation equilibrium constant ( K ) for cyanide binding to Mb(III) ( k off = k on  ×  K ).