These studies and others support a mechanism whereby an exchange-labile Cou(phen)x complex is rapidly and reversibly chelated by SF,. The Con(phen)x-enzyme complex is oxidized in situ by [Com(phen)2C03]+ to yield an exchange-inert ConI(phen)x-SF, complex with no ATPase activity. Inactivations of SF, which result from Con(phen)x in situ oxidation by either H202 or 02 are not reversed by reducing agents, despite the removal of bound cobalt. These data indicate in situ oxidation of CoII(phen)x by the single electron-accepting oxidant [ConI(phen)2C03]+ results in a very mild incorporation of Co(III) into SF,. This approach may have general application for the incorporation of Co(III) into proteins.agents which convert the exchange-inert cobalt(III) back to exchange labile Co(II).
This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.
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