Molybdenum Cofactor from Tobacco Cell Cultures and Milk Xanthine Oxidase: Involvement of Sulfhydryl Groups in Dimerization Activity of Cofactor

Abstract

Molybdenum cofactor isolated by heat treatment from cell cultures of Nicotiana tabacum and from milk xanthine oxidase, is shown to be efficiently stabilized by reduced glutathione. Activity of molybdenum cofactor was determined by restoring nitrate reductase activity in extracts of Neurospora crassa mutant nit-1. The results obtained suggest the involvement of sulfhydryl groups in the cofactor-mediated processes of subunit dimerization to form active nitrate reductase in N. crassa nit-1, as well as in the correct liganding of molybdenum in nitrate reductase. Both these processes can be blocked by p-hydroxymercuribenzoate. This report also describes a novel method for producing and detecting of catalytically defective molybdenum cofactor from extracts of tobacco cell cultures and from milk xanthine oxidase. Two forms of defective molybdenum cofactor are shown to occur: molybdenum-free form A of cofactor is still able to effect dimerization of nitrate reductase while form B is more severely impaired since it binds to solely one nitrate reductase subunit without being able to mediate subunit assembly. Form B is insensitive to oxygen at room temperature. The involvement of disulfide bridges in linking the subunits in the dimeric holoform of nitrate reductase, will be discussed in detail.