| Abstract
| - The electron-transfer reaction between reduced cobalamin (Cbl(II)) and sodium pentacyanonitrosylferrate(II) (sodium nitroprusside, NP), as well as the subsequent processes following the electron-transfer step, were investigated by spectroscopic (UV−vis, 1H NMR, EPR), electrochemical (CV, DPV)and kinetic (stopped-flow) techniques. In an effort to clarify the complex reaction pattern observed atphysiological pH, systematic spectroscopic and kinetic studies were undertaken as a function of pH (1.8−9) and NP concentration (0.0001 − 0.09 M). The kinetics of the electron-transfer reaction was studiedunder pseudo-first-order conditions with respect to NP. The reaction occurs in two parallel paths of differentorder, viz. pseudo-first and pseudo-second order with respect to the NP concentration, respectively. Thecontribution of each path depends on pH and the [NP]/[Cbl(II)] ratio. At low pH and total NP concentration(pH < 3, [NP]/[Cbl(II)] ≈ 1), the cyano-bridged successor complex [Cbl(III)−(μ-NC)-FeI(CN)3(NO+)]- (1s) isthe final reaction product formed in an inner-sphere electron transfer reaction that is coupled to the releaseof cyanide from coordinated nitroprusside. At higher pH, subsequent reactions were observed which involvethe attack of cyanide released in the electron transfer step on the initially formed cyano-bridged species,and lead to the formation of Cbl(III)CN and [FeI(CN)4(NO+)]2-. The strong dependence of the rate andmechanism of the subsequent reactions on pH is attributed to the large variation in the effective nucleophilicityof the cyanide ligand in the studied pH range. An alternative electron-transfer pathway observed in thepresence of excess NP involves the reaction of the precursor complex [Cbl(II)−(μ-NC)−FeII(CN)4(NO+)]2-(1p) with NP to give [Cbl(III)−(μ-NC)−FeII(CN)4(NO+)]- (2) and reduced nitroprusside, [Fe(CN)5NO]3-, asthe initial reaction products. Analysis of the kinetic data allowed elucidation of the rate constants for theinner- and outer-sphere electron-transfer pathways. The main factors which influence the kinetics andthermodynamics of the observed electron-transfer steps are discussed on the basis of the spectroscopic,kinetic and electrochemical results. A general picture of the reaction pathways that occur on a short (s)and long (min to h) time scale as a function of pH and relative reactant concentrations is derived from theexperimental data. In addition, the release of NO resulting from the one-electron reduction of NP by Cbl(II)was monitored with the use of a sensitive NO electrode. The results obtained in the present study arediscussed in reference to the possible influence of cobalamin on the pharmacological action of nitroprusside.
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