Crystallographic determination of wild type, mutant and substrate-analogue inhibited structures of bacterial members of a family of superoxide dismutases : submitted as part of the requirements for the degree of Doctor of Philosophy, Institute of Fundamental Sciences, Chemistry, Massey University, New Zealand
The iron and manganese superoxide dismutases are a family of metallo-enzymes
with highly conserved protein folds, active sites and dimer interfaces. They
catalyse the elimination of the cytotoxic free radical superoxide to molecular
oxygen and hydrogen peroxide by alternate reduction then oxidation of the activesite
with the concomitant transfer of protons from the solvent. There are many key
aspects of enzymatic function that lack a structural explanation.
The focus of this study is on three crystal structures. The iron-substituted
manganese superoxide dismutase from Escherichia coli complexed with azide, a
substrate-mimicking inhibitor, was solved to 2.2 Å. This “wrong” metal form
shows a binding pattern seen previously in the manganese superoxide dismutase
from Thermus thermophilus. Wild-type manganese specific superoxide dismutase
from the extremophile Deinococcus radiodurans was solved to 2.0 Å and has an
active site reminiscent of other solved manganese superoxide dismutases despite a
lack of product inhibition. The azide-inhibited manganese superoxide dismutase
from Deinococcus radiodurans was determined to a resolution of 2.0 Å and
showed binding of azide, and by inference superoxide, different to that seen in
Thermus thermophilus, but reminiscent of that seen in azide-inhibited iron
superoxide dismutases. These results indicate that the azide ion, and by inference
superoxide, bind to the metal centre of manganese superoxide dismutases in two
modes, and transition between the two modes may be entropy dependent.
These structures, integrated with knowledge from other structures, known
biochemistry and various spectra, provide insight into catalytic function. An
outer-sphere mechanism of proton transfer that does not rely on through-peptide
proton uptake is proposed and compared to a previously proposed inner-sphere
mechanism. This is based on the observation that a water molecule moves into the
active site of the manganese superoxide dismutase from Deinococcus radiodurans
upon azide binding, providing a Grötthus pathway for rapid proton transfer to the
active site from the bulk solvent.
Also presented in this study are the partially refined structures of four point
mutants (S82T, L83M, L133V, and M164L/L166V) of the manganese superoxide
dismutase from Escherichia coli all solved to roughly 2 Å resolution, designed to
investigate product inhibition which varies across species.