Identifiction and assay of synchrotron radiation‐induced alterations on metalloenzymes and proteins

In pulsed radiolysis studies, 4 components of the respiratory chain have been demonstrated to accept electrons from the products of the interaction of ionizing radiation and water molecules: cytochrome c, a heme protein electron-transfer component [ 11; ubiquinone, an electron-transfer component [2]; riboflavin (and presumably FAD and FMN), an eiectron-acceptor from substrates [3]; and NAD, the principal redox agent of the citric acid cycle in the mitochondriai respiratory chain [4]. In the last 3 cases, the 2 eredox mediators are reduced to the semiquinone, while in cytochrome c, Fe3’ is reduced to Fe’*. The reactions are extremely rapid: rate constants approaching the diffusion-limited values of 10” M-’ s-’ are obtained at pl~ysiological pH values in the case of cytochrome c. In fact, hydrated electrons will radicalize nearly any organic substance, which in turn can react rapidly with cytochronle c, causing its reduction [ 11. In pulse radiolysis studies of cytochrome oxidase, 2.45 PM hydrated electrons are produced of which -50% or 1 .15 PM react with 1 .15 PM cytochrome oxidase in a half-time of 3.7 ps [5]. In synchrotron radiation studies, usual beam intensities at the sample (-6 X 10” pIlotons~s) are calculated to give -2.5 PM hydrated electrons/s. Thus, a similar, or even more efficient, reaction may occur with the I mM cytochrome oxidase in the 0.5-l h X-irradiation required for adequate accuracy of data collection. Since a variety of metaIloproteins have been employed in wide ranging studies [G-9], with similar reactions possibly occurring, it seems useful to report here our studies in which the redox state of the metalloproteins under irradiation could