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.
University of Minnesota Ph.D. dissertation. April 2010. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: John D. Lipscomb. 1 computer file (PDF); xi, 214 pages. Ill. (som col.)
Insight into the oxygen activation mechanism by Rieske dioxygenases through kinetic, spectroscopic and mutagenesis studies..
Retrieved from the University of Minnesota Digital Conservancy,
Content distributed via the University of Minnesota's Digital Conservancy may be subject to additional license and use restrictions applied by the depositor.
Showing items related by title, author, creator and subject.
Homoprotocatechuate (HPCA) 2,3-dioxygenase (HPCD) is an Fe(II)-dependent extradiol-cleaving dioxygenase, which oxidatively cleaves the aromatic C(2)-C(3) bond of its catecholic substrate. Here we compare the reactivity of ...
Hematopoietic stem cell transplant is the only curative therapy for a variety of malignant and non-malignant hematologic disorders and has the potential to treat a variety of other difficult diseases. Unfortunately, the ...
Homoprotocatechuate 2,3-Dioxygenase (2,3-HPCD) from Brevibacterium fuscum
catalyzes the O2-dependent extradiol ring-opening reaction of its catecholic
substrate homoprotocatechuate (HPCA) to yield a α-hydroxy-δ-carboxy ...