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Browsing by Subject "Kinetic and Spectroscopic Studies of Rieske Dioxygenase"

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    Insight into the oxygen activation mechanism by Rieske dioxygenases through kinetic, spectroscopic and mutagenesis studies.
    (2010-04) Chakrabarty, Sarmistha
    Rieske dioxygenases catalyze the first step in the degradation of aromatic hydrocarbons. They facilitate dioxygen bond cleavage with insertion of both O atoms as hydroxyl groups into the aromatic substrate; this produces a non-aromatic cis-diol. Here studies of the chemical and regulatory mechanisms of benzoate 1,2-dioxygenase (BZDO) and naphthalene 1,2-dioxygenase (NDO) are described. These multicomponent enzymes consist of an (αβ)3 oxygenase component in addition to a reductase and, in the case of NDO, ferredoxin components that mediate the electron transfer from NAD(P)H. The oxygenase component contains a Rieske [2Fe-2S] cluster and a non-heme mononuclear Fe center, which is the site for the O2 activation and product formation. Transient kinetic and spectroscopic studies of BZDO show that electron transfer from the Rieske cluster to an adjacent Fe center across the subunit boundary occurs in 3 phases due to the presence of at least 2 and probably 3 different types of active sites. These differences in nominally identical active sites are proposed to originate from structural changes related to redox state-mediated regulation. This is demonstrated by a Magnetic Circular Dichroism study with NDO that reveals changes in iron coordination number and geometry controlled by the redox state of the Rieske cluster and the presence of substrate. Mutagenesis studies of the essential subunit interface residue Asp205 in NDO show that it is unlikely to be the sole mediator of electron transfer and regulatory conformational change as proposed by others. The nature of the reactive oxygen intermediate formed at the Fe site was probed using the radical clock substrate probes norcarane and bicyclohexane. They show that monooxygenase chemistry by NDO occurs via a substrate radical, implicating formation of a novel HO-Fe5+=O reactive state that may also pertain to dioxygenase chemistry.