Choline oxidase serves as a paradigm for alcohol oxidation catalyzed by flavin-dependent enzymes. In its active site, S101 is 4 Å from the flavin C4a atom on an extended loop. Enzyme variants substituted at S101 were generated in a previous study and investigated mechanistically [Yuan, H., and Gadda, G. (2011) Biochemistry 50, 770-779]. In this study, the typical ultraviolet-visible (UV-vis) absorption spectrum of oxidized flavin was observed for the S101C enzyme in HEPES, TES, or sodium phosphate, whereas an absorption spectrum suggesting the presence of a C4a-flavin adduct with cysteine was obtained in Tris-HCl at pH 8.0. pH titrations of the UV-vis absorption spectrum of the wild-type, S101A, S101C, and H99N enzymes in the presence and absence of Tris allowed for the determination of two pKa values that define a pH range in which the C4a-S-cysteinyl flavin is stabilized. Inhibition studies and stopped-flow kinetics demonstrated that binding of protonated Tris in the active site of the S101C enzyme is required to form the C4a-S-cysteinyl flavin. Deuterium kinetic isotope effects and proton inventories of the S101C enzyme mixed in a stopped-flow spectrophotometer with Tris established a mechanism for the reversible formation of the C4a-S-cysteinyl flavin. This study provides a detailed mechanistic analysis of the reversible formation of a bicovalent C4a-S-cysteinyl-8α-N3-histidyl flavin in choline oxidase, identifying an optimal pH range and a mechanistic rationale for the stabilization of de novo C4a-S-cysteinyl-flavins. Moreover, it presents an example of an intramolecular reaction of an enzyme-bound flavin without a substrate.