||<p>Sulfite oxidase, a metabolically important enzyme, catalyzes the physiologically
critical conversion of sulfite to sulfate in the terminal step of the degradation
of sulfur containing compounds. The enzyme has been the focus for much research since
its discovery in the 1950's. A central question to understanding the mechanism of
molybdoenzymes such as sulfite oxidase and nitrate reductase concerns the roles of
active site residues and the coordination chemistry of the Mo atom in the structure
and function of the enzyme. The goal of this work was directed towards the characterization
and determination of the structures of active site variants of sulfite oxidase using
a spectroscopic, kinetic, and protein crystallographic approach.</p><p>Earlier studies
have identified a single, highly conserved cysteine residue as the donor of a covalent
bond from the protein to molybdenum in sulfite oxidase and nitrate reductase. The
C185S and C185A variants of chicken sulfite oxidase exhibited severely attenuated
activity. Crystallographic and spectroscopic analysis of both variants revealed a
change in the metal coordination, from a dioxo to a trioxo form of Mo. </p><p>Assimilatory
nitrate reductase is a member of the sulfite oxidase family of molybdopterin enzymes.
The crystal structure of the Mo domain of the enzyme from Pichia angusta revealed
high structural homology in the active sites of nitrate reductase and sulfite oxidase.
Both enzymes utilize the same form of the molybdenum cofactor and have three out of
five residues conserved at the active site. Substitution of two active site residues
in sulfite oxidase alters the substrate affinity of chicken SO from sulfite to nitrate,
resulting in an increase of nitrate reductase activity over wild-type sulfite oxidase.
Additionally we identified an additional amino acid position in sulfite oxidase that
corresponds to a non-conserved position in NR that further increased NR activity.
Finally, these nitrate reductase variants of sulfite oxidase were crystallized and
the structures solved. This represents the first example of the transmutation of
a molybdenum enzyme to change activity and substrate affinity to those of a homologous