Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale.

Center for Rare Diseases Hospital Universitario de Canarias Universidad de la Laguna 38320 Tenerife Spain

Departamento de Bioquímica y Biología Molecular y Cellular Facultad de Ciencias Instituto de Biocomputación y Física de Sistemas Complejos Universidad de Zaragoza 50009 Zaragoza Spain

Departamento de Química Física Unidad de Excelencia en Química Aplicada a Biomedicina y Medioambiente e Instituto de Biotecnología Universidad de Granada Av Fuentenueva s n 18071 Granada Spain

Departamento de Química Física Universidad de Granada Av Fuentenueva s n 18071 Granada Spain

Department of Biochemistry and Molecular Biology 1 Faculty of Sciences and Biomedical Research Center University of Granada Granada Spain

Institute of Biotechnology BioCeV Academy of Sciences of the Czech Republic Prumyslova 595 Vestec 252 50 Czech Republic

Institute of Microbiology BioCeV Academy of Sciences of the Czech Republic Prumyslova 595 Vestec 252 50 Czech Republic

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