ACTIVE SITES OF FREE RADICAL METALLOENZYMES

  • Whittaker, James (PI)

Project: Research project

Project Details

Description

Free radical biochemistry is an important and emerging new area probing
the involvement of organic free radicals, molecules having unpaired
electrons, in biochemical processes. Historically, studies on free
radicals in biology have emphasized the deleterious effects of radicals
in toxicity mechanisms and radiation damage, but there is a growing
recognition of the essential role that free radicals play in enzyme
catalysis and the biological function of redox metalloproteins. Early
studies in the area of free radical enzymology focussed on Fe- and B12-
dependent ribonucleotide reductases and mutases from certain anaerobic
bacteria. However, the significance of radicals in biochemical
mechanisms is now well established in enzymes with as important and
diverse functions as prostaglandin and penicillin biosynthesis, lipid
peroxidation and alkane oxidation. A motif which is emerging from these
studies is the association of the radical site with a metal center that
is involved in generation, stabilization or control of reactivity of
radicals in proteins. Two of the most well-defined free radical
metalloenzymes are galactose oxidase and the Fe-dependent ribonucleotide
reductase RRB2. Each of these enzymes stabilize a protein side chain as
a free radical in the resting enzyme, a radical which is required for
catalytic activity. Extensive biochemical and basic spectroscopic
characterization is now complete for both proteins, and both are also
crystallographically defined at near atomic resolution. Detailed
spectroscopic studies of these proteins can now be expected to extend to
geometric information available in the crystalligraphic data to develop
the special electronic structural features of the radical sites and
metal interactions, leading to insights into the mechanism of
stabilization of radicals in proteins and the control of their unique
reactivity. these key aspects of free radical biochemistry have not been
previously defined by protein structural studies. These key aspects of
free radical biochemistry have not been previously defined by protein
structural studies. Our studies will focus on the information contained
in electron paramagnetic resonance (EPR), electronic absorption,
circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of
the active site complexes in galactose oxidase (GO), ribonucleotide
reductase (RRB2) and isopenicillin N synthase (IPNS). In the former (GO,
RRB2) we will probe the radical sites as well as the biological metal
complex. For IPNS the resting ferrous enzyme and its substrate complex
will be of key interest in terms of radical-generating species. In all
three studies, inorganic models will provide important calibration. To
probe the mechanisms of reactivity for free radical metalloenzymes we
will explore chemical perturbations, examining the spectroscopic and
electrochemical consequences of coordinating exogenous ligands in the
active complexes, and investigate the details of turnover reaction using
transient kinetics methods. These fundamental studies will form the
basis for characterization of other important and interesting free
radical metalloenzymes.
StatusFinished
Effective start/end date2/1/928/31/10

Funding

  • National Institutes of Health: $236,432.00
  • National Institutes of Health: $247,034.00
  • National Institutes of Health
  • National Institutes of Health: $199,633.00
  • National Institutes of Health: $205,000.00
  • National Institutes of Health: $205,000.00
  • National Institutes of Health: $205,522.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $199,212.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $205,000.00

ASJC

  • Medicine(all)
  • Biochemistry, Genetics and Molecular Biology(all)

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