The final step of the biosynthesis of prokaryotic cofactor PQQ is catalyzed by PqqC, a cofactorless oxidase that brings about a ring closure and overall eight-electron oxidation of its substrate. Time-dependent acid quenching and subsequent high-performance liquid chromatography separation and mass spectrometric analyses of reaction mixtures were performed to correlate the structures of intermediates with previously observed UV-visible signatures. The reaction is composed of four stepwise oxidations: three steps use O2 as the two-electron acceptor, and the fourth uses hydrogen peroxide (H2O2). The chemical nature of the intermediates, the stoichiometry of the reaction, and their dependence on the oxygen concentration indicate that the third oxidation uses the product, H2O2, from the preceding step to produce water. The last oxidation step can also be studied separately and is a reaction between O2 and PQQH2 trapped in the active site. This oxidation is approximately 10 times slower than the reoxidation of PQQH2 in solution. From the order of the four oxidation steps and their sensitivity to O2 concentration, we propose a progressive closure of the active site as the enzyme proceeds through its catalytic cycle.