Thioredoxin reductase (TrxR) is one class of the most important
antioxidant selenoproteins and is involved in regulating
tumor genesis and progression. It has been reported that
naphthoquinones can target and inhibit TrxR1 activity therefore produce
reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the
naphthoquinone compounds to become potential antitumor
chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and
menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated
naphthoquinone reduction and inhibition of TrxR1 by
naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of
menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of
menadione on TrxR1 activity, and eventually identified the interaction between
menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated
menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the
menadione reduction, indicating that TrxR1-mediated
menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for
menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the
menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of
menadione, suggesting that
menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of
selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of
menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by
menadione. Hereby it will be useful for the research and development of
naphthoquinone anticancer drugs targeting TrxR1.