Electron transfer kinetics of the mitochondrial outer membrane protein mitoNEET

Xiaokang Li, Wenzhou Medical University
Yiming Wang, Louisiana State University
Guoqiang Tan, Wenzhou Medical University
Jianxin Lyu, Wenzhou Medical University
Huangen Ding, Louisiana State University

Abstract

© 2018 Elsevier Inc. Increasing evidence suggests that the mitochondrial outer membrane protein mitoNEET is a key regulator of energy metabolism, iron homeostasis, and production of reactive oxygen species in mitochondria. Previously, we reported that mitoNEET is a redox enzyme that catalyzes electron transfer from the reduced flavin mononucleotide (FMNH 2 ) to oxygen or ubiquinone via its unique [2Fe-2S] clusters. Here, we explore the reduction and oxidation kinetics of the mitoNEET [2Fe-2S] clusters under anaerobic and aerobic conditions. We find that the mitoNEET [2Fe-2S] clusters are rapidly reduced by a catalytic amount of FMNH 2 which is reduced by flavin reductase and an equivalent amount of NADH under anaerobic conditions. When the reduced mitoNEET [2Fe-2S] clusters are exposed to air, the [2Fe-2S] clusters are slowly oxidized by oxygen at a rate constant of about 6.0 M −1 s −1 . Compared with oxygen, ubiquinone-2 has a much higher activity to oxidize the reduced mitoNEET [2Fe-2S] clusters at a rate constant of about 3.0 × 10 3 M −1 s −1 under anaerobic conditions. Under aerobic conditions, the mitoNEET [2Fe-2S] clusters can still be reduced by FMNH 2 in the presence of flavin reductase and excess NADH. However, when NADH is completely consumed, the reduced mitoNEET [2Fe-2S] clusters are gradually oxidized by oxygen. Addition of ubiquinone-2 also rapidly oxidizes the pre-reduced mitoNEET [2Fe-2S] clusters and effectively prevents the FMNH 2 -mediated reduction of the mitoNEET [2Fe-2S] clusters under aerobic conditions. The results suggest that ubiquinone may act as an intrinsic oxidant of the reduced mitoNEET [2Fe-2S] clusters in mitochondria under aerobic and anaerobic conditions.