Kinetic and mechanistic studies of heterotetrameric sarcosine oxidase from Arthrobacter sp.1-IN

Heterotetrameric sarcosine oxidase (TSOX; EC. 1.5.3.1) from Arthrobacter sp. 1-IN is a complex flavoprotein that catalyses the oxidative demethylation of sarcosine to produce glycine, formaldehyde and hydrogen peroxide. TSOX is composed of four subunits with approximate molecular weights of 106, 43, 24 and 15 kDa the genes for which have been cloned and the recombinant protein expressed at low levels.;The Arthrobacter sp. 1-IN genomic DNA containing the sox operon was sequenced and, as well as the four TSOX genes, contains genes for a serine hydroxylmethyltransferase and a formyl-tetrahydrofolate deformylase. Native and recombinant TSOX were expressed, purified and characterised. Cofactor analysis showed that TSOX contains 1 mol each of non-covalently bound FAD and NAD+ and also 1 mol of covalent flavin.;The reductive half-reaction of TSOX was studied using stopped-flow spectroscopy. pH dependence of flavin reduction by sarcosine indicated no kinetically influential ionisations in the enzyme-substrate complex. Two ionisations with pKa values of 7.4 0.1 and 10.4 0.2 were identified from a klim/Kd versus pH plot. Kinetic isotope effects studies of the rate of C-H bond breakage in sarcosine indicated a ground-state quantum tunnelling mechanism for H-transfer assisted by the thermal fluctuations of the protein molecule.;Anaerobic reduction experiments indicated that the two flavins of TSOX were reducible and that two electrons were required per flavin for complete reduction. The redox potentials for the two flavins were determined and indicated that one flavin had an unusually high redox potential. The inter-flavin electron transfer rate was measured using a pH jump technique between pH 7 and 9. The pH jump experiments and redox potential measurements indicated that the intramolecular electron transfer in TSOX was an endergonic process.