Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.

Biomedical Research Centre Slovak Academy of Sciences Dúbravská cesta 9 845 05 Bratislava Slovakia

Centre for Interdisciplinary Biosciences P J Šafárik University in Košice Jesenná 5 04154 Košice Slovakia

Department of Biochemistry Faculty of Science P J Šafárik University in Košice Moyzesova 11 04154 Košice Slovakia

Department of Biochemistry University of Zurich Winterthurerstrasse 190 CH 8057 Zurich Switzerland

Department of Biophysics P J Šafárik University in Košice Jesenná 5 04154 Košice Slovakia

Institute of Microbiology of the CAS v v i Videnska 1083 142 20 Prague 4 Czech Republic

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