Reductive nitrosylation of ferric cyanide horse heart myoglobin is limited by cyanide dissociation |
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Authors: | Paolo Ascenzi Alessandra di Masi Marco Mattu Massimo Coletta |
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Affiliation: | a Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘Roma Tre’, Viale Guglielmo Marconi 446, I-00146 Roma, Italy b Department of Experimental Medicine and Biochemical Sciences, University of Roma ‘Tor Vergata’, Via Montpellier 1, I-00133 Roma, Italy c Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems (CIRCMSB), Piazza Umberto I 1, I-87100 Bari, Italy |
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Abstract: | Cyanide binds to ferric heme-proteins with a very high affinity, reflecting the very low dissociation rate constant (koff). Since no techniques are available to estimate koff, we report herewith a method to determine koff based on the irreversible reductive nitrosylation reaction to trap ferric myoglobin (Mb(III)). The koff 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 koff value, representing the rate-limiting step, can be directly determined. The koff value obtained experimentally matches very well to that calculated from values of the second-order rate constant (kon) and of the dissociation equilibrium constant (K) for cyanide binding to Mb(III) (koff = kon × K). |
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Keywords: | Ferric cyanide horse heart myoglobin Nitrogen monoxide Reductive nitrosylation Kinetics |
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