Sulfite : Cytochrome c oxidoreductase from Thiobacillus novellus - Purification, characterization, and molecular biology of a heterodimeric member ofthe sulfite oxidase family

Direct oxidation of sulfite to sulfate occurs in various photo- and chemotr ophic sulfur oxidizing microorganisms as the final step in the oxidation of reduced sulfur compounds and is catalyzed by sulfite:cytochrome c oxidored uctase (EC 1.8.2.1), Here we show that the enzyme from Thiobacillus novellu s is a periplasmically located alpha beta heterodimer, consisting of a 40.6 -kDa subunit containing a molybdenum cofactor and an 8.8-kDa monoheme cytoc hrome c(552) smbunit (midpoint redox potential, Em(8.0) = +280 mV), The org anic component of the molybdenum cofactor was identified as molybdopterin c ontained in a 1:1 ratio to the Mo content of the enzyme. Electron paramagne tic resonance spectroscopy revealed the presence of a sulfite-inducible Mo( V) signal characteristic of sulfite:acceptor oxidoreductases. However, pH-d ependent changes in the electron paramagnetic resonance signal were not det ected. Kinetic studies showed that the enzyme exhibits a ping-pong mechanis m involving two reactive sites. K-m values for sulfite and cytochrome c(550 ) were determined to be 27 and 4 mu M, respectively; the enzyme was found t o be reversibly inhibited by sulfate and various buffer ions. The sorAB gen es, which encode the enzyme, appear to form an operon, which is preceded by a putative extracytoplasmic function-type promoter and contains a hairpin loop termination structure downstream of sorB. While SorA exhibits signific ant similarities to known sequences of eukaryotic and bacterial sulfite:acc eptor oxidoreductases, SorB does not appear to be closely related to any kn own c-type cytochromes.