The Mo(V) center of plant sulfite oxidase from
Arabidopsis thaliana (At-SO) has been studiedby continuous wave and pulsed EPR methods. Three different Mo(V) EPR signals have been observed,depending on pH and the technique used to generate the Mo(V) oxidation state. At pH 6, reduction bysulfite followed by partial reoxidation with ferricyanide generates an EPR spectrum with
g-values similarto the low-pH (
lpH) form of vertebrate SOs, but
no nearby exchangeable protons can be detected. On theother hand, reduction of At-SO with Ti(III) citrate at pH 6 generates a Mo(V) signal with large hyperfinesplittings from a single exchangeable proton, as is typically observed for
lpH SO from vertebrates. Reductionof At-SO with sulfite at high pH generates the well-known high-pH (
hpH) signal common to all sulfiteoxidizing enzymes. It is proposed that, depending on the conformation of Arg374, the active site ofAt-SO may be in "closed" or "open" forms that differ in the degree of accessibility of the Mo center tosubstrate and water molecules. It is suggested that at low pH the sulfite-reduced At-SO has coordinatedsulfate and is in the "closed form". Reoxidation to Mo(V) by ferricyanide leaves bound sulfate trappedat the active site, and consequently, there are no ligands with exchangeable protons. Reduction withTi(III) citrate injects an electron directly into the active site to generate the [Mo
V
O(OH)]
2+ unit that iswell-known from model chemistry and which has a single exchangeable proton with a large isotropichyperfine interaction. At high pH, the active site is in the "open form", and water can readily exchangeinto the site to generate the
hpH SO.
