Key Role of Alkanoic Acids on the Spectral Properties, Activity, and Active‐Site Stability of Iron‐Containing Nitrile Hydratase from Brevibacterium R312

Abstract

Interaction of n-butyric acid with dialyzed nitrile hydratase from Brevibacterium R312, which is characterized by a charge-transfer band at 680 nm and EPR signals typical of a low-spin Fe(III) with delta g = 0.22, leads to a form displaying different spectral properties (lambda = 710 nm, delta g = 0.31). Butyric acid also acts as a competitive inhibitor of nitrile-hydratase-catalyzed hydration of acrylonitrile with a Ki value of 0.9 mM. Formation of the complex between the enzyme and butyric acid is highly dependent on the concentration of the latter and on pH. When stored with high levels of butyric acid, nitrile hydratase is completely inactive. The active uncomplexed enzyme is restored under the high dilution conditions used for the enzymatic assays, while the complexed form is favored at acidic pH and is not formed at pH above 8. Furthermore, the inhibitory potency of butyric acid decreases upon increasing pH (IC50 increases from 0.8 mM at pH 6.2 to 12 mM at pH 8.2). These data show that nitrile hydratase interacts with the acid form of butyric acid with a high affinity (Ki' approximately 4 microM at pH 7.2). At pH < 3, the visible spectrum of the enzyme disappears, presumably because of demetallation, whereas that of the complex exhibits a charge-transfer band shifted to 800 nm, the presence of butyric acid preventing nitrile hydratase from demetallation. Other linear carboxylic acids such as valeric and hexanoic acids behave similarly; they act as inhibitors of nitrile hydratase and protect the enzyme during storage. A structure of the nitrile hydratase active site interacting with butyric acid is tentatively proposed in which the latter is hydrogen-bonded to the Fe(III)-OH moiety. This interaction between butyric acid and nitrile hydratase should be considered when deducing the nature of nitrile hydratase active site and mechanisms, from spectral and enzymatic data, since most results published previously have been obtained on nitrile hydratase containing large amounts of butyric acid and interpreted without taking into account the presence of this acid in the active site.