The large and multifunctional class IV E2 enzyme UBE2O, comprising 1,292 amino acids, plays a crucial role in orchestrating ubiquitin transfer from the E1 enzyme UBA1 and facilitating substrate ubiquitination through its distinctive hybrid E2/E3 functionality. Using cryoEM, we have resolved a series of conformational intermediates of the human UBA1-UBE2O complex, revealing a release-and-go regulatory mechanism. We demonstrate that upon UBA1 interaction, the N-terminal domains of UBE2O (UBE2O-N) are released without further engagement with either UBA1 or UBE2O itself. Furthermore, we have uncovered the short insertion loop B within UBE2O, which shields or exposes the catalytic Cys1040, acting as a molecular switch to regulate transthiolation. Beyond these mechanistic insights, we have introduced AI-designed miniproteins that promotes the release of UBE2O-N and consequently enhance transthiolation from UBA1 to UBE2O allosterically and stimulate the multi-monoubiquitination of BAP1. These findings not only elucidate the structural and functional principles governing the E2-E3 hybrid activity of UBE2O but also open new avenues for the precision modulation of ubiquitin signaling pathways. Our work redefines the mechanisms by the E2/E3 hybrid UBE2O achieve specificity and control, paving the way for future therapeutic strategies targeting ubiquitin-related pathways.