Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation.
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 degrees 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)xK).
Ascenzi
Paolo
P
Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University 'Roma Tre', Roma, Italy. ascenzi@uniroma3.it
di Masi
Alessandra
A
Gullotta
Francesca
F
Mattu
Marco
M
Ciaccio
Chiara
C
Coletta
Massimo
M
D000818:Animals; D005292:Ferricyanides / Q000737:chemistry / Q000378:metabolism*; D006454:Hemoglobins / Q000737:chemistry / Q000378:metabolism*; D006736:Horses; D008786:Metmyoglobin / Q000737:chemistry / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D010084:Oxidation-Reduction
D005292:Ferricyanides; D006454:Hemoglobins; C007177:cyanometmyoglobin; C008657:nitrosyl hemoglobin; D008786:Metmyoglobin; D009569:Nitric Oxide