Abstract
The oligomerization of the flavoprotein vanillyl-alcohol oxidase (VAO) and its site-directed mutant H61T was studied by mass spectrometry. Native VAO has a covalently bound FAD and forms primarily octameric assemblies of 507 kDa. H61T is purified as a FAD-free apoprotein and mainly exists as a dimeric species of 126 kDa. Binding of FAD to apoH61T rapidly restores enzyme activity and induces octamerization, although association of H61T dimers seems not to be crucial for enzyme activity. Reconstitution of H61T with the cofactor analog 5'-ADP also promotes octamerization. FMN on the other hand, interacts with apoH61T without stimulating dimer association. These results are in line with observations made for several other flavoenzymes, which contain a Rossmann fold. Members of the VAO flavoprotein family do not contain a Rossmann fold but do share two conserved loops that are responsible for binding the pyrophosphate moiety of FAD. Therefore, the observed FAD-induced oligomerization might be general for this family. We speculate that upon FAD binding, small conformational changes in the ADP-binding pocket of the dimeric VAO species are transmitted to the protein surface, promoting oligomerization.
Highlights
Riboflavin derivatives such as FAD and FMN are essential components of all living organisms, serving as cofactors for numerous proteins with diverse functions, ranging from electron transport, redox catalysis, oxygen activation, and light emission to DNA repair [1]
It is well established that mass spectrometry may be used to detect even weakly bound noncovalent complexes of protein assemblies, even when they are very large and high in mass as the ones described here
We have been able to detect ions in the mass spectra corresponding to the dimeric assembly as well as the octameric assembly of the protein Vanillyl-alcohol oxidase (VAO)
Summary
Riboflavin (vitamin B2) derivatives such as FAD and FMN are essential components of all living organisms, serving as cofactors for numerous proteins with diverse functions, ranging from electron transport, redox catalysis, oxygen activation, and light emission to DNA repair [1]. The flavin cofactor is noncovalently bound, ϳ10% of human cellular FAD is covalently linked to enzymes like monoamine oxidase and succinate dehydrogenase [2, 3]. The functional role of the covalent protein-flavin linkage in VAO has been addressed recently [26] by site-directed mutagenesis. From the properties of His-422 mutants, evidence was obtained that the covalent linkage between His-422 and the apoprotein is important for VAO catalysis by raising the redox potential of the flavin. All three mutants H422A, H422C, and H422T tightly bind the FAD in a noncovalent mode, but the change in redox properties results in a marked decrease in the rate of substrate-mediated flavin reduction
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