Abstract
The attenuation of the sulfite:cytochrome c activity of sulfite oxidase upon treatment with ferricyanide was demonstrated to be the result of oxidation of the pterin ring of the molybdenum cofactor in the enzyme. Oxidation of molybdopterin (MPT) was detected in several ways. Ferricyanide treatment not only abolished the ability of sulfite oxidase to serve as a source of MPT to reconstitute the aponitrate reductase in extracts of the Neurospora crassa mutant nit-1 but also eliminated the ability of sulfite oxidase to reduce dichlorobenzenoneindophenol after anaerobic denaturation. Additionally, the absorption spectrum of anaerobically denatured ferricyanide-treated molybdenum fragment of rat liver sulfite oxidase was typical of fully oxidized pterins. Ferricyanide treatment had no effect on the protein of sulfite oxidase or on the sulfhydryl-containing side chain of MPT. Quantitation of the ferricyanide reaction showed that 2 mol of ferricyanide were reduced per mol of MPT oxidized, yielding a fully oxidized pterin. These results corroborate the previously reported conclusion that the native state of reduction of MPT in sulfite oxidase is at the dihydro level (Gardlik, S., and Rajagopalan, K.V. (1990) J. Biol. Chem. 265, 13047-13054). As a result of oxidation of the pterin ring, the affinity of MPT for molybdenum is decreased, leading to eventual loss of molybdenum. Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT. These molecules retain sulfite:O2 activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased sulfite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observations suggest that the pterin ring of MPT participates in enzyme function, possibly in electron transfer, directly in catalysis, or by controlling the oxidation/reduction potential of molybdenum.
Highlights
The attenuation of the su1fite:cytochrome c activity nal amplitude
They postulated that a nonprotein ligand of the molybdenum might be involved in mediating electron transfer from the molybdenum to theone-electron acceptors of the enzyme and that this ligand could be the site of oxidative modification by ferricyanide
We report the results of several studies which serve to clarify the mechanism of inactivation by ferricyanide and toprovide evidence that the pterin of the molybdenum cofactor isinvolved in molybdoenzyme function
Summary
Because the loss of molybdenum is slow, a population of sulfite oxidase molecules can exist in which molybdenum is complexed to oxidized MPT These molecules retain sulfite: activity, a function apparently dependent solely on the molybdenum-thiolate complex, yet have greatly decreased su1fite:cytochrome c activity, a function requiring heme as well as the molybdenum center of holoenzyme. These observationssuggest that the pterin roinf gMPT bic denaturation of ferricyanide-treated sulfite oxidase, followed by gel filtration and QAE-Sephadex chromatography; the conclusion that MPT exists in the enzyme as the form isolated could be questioned.
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