Catalyzes the oxidation of glycolate to glyoxylate, with a reduction of O2 to H2O2 (PubMed:25416784). Is an essential enzyme in photorespiration in plants (PubMed:18805949). Photorespiration plays a vital role in C4 photosynthesis in Z.mays and is essential for maize seedling development and maintaining low (non-toxic) levels of glycolate (PubMed:18805949). glycolate + O2 = glyoxylate + H2O2 Binds 1 FMN per subunit. kcat is 34.13 sec(-1) (at pH 8 and 30 degrees Celsius). Photosynthesis; photorespiration; glycine from 2-phosphoglycolate: step 2/3. Homotetramer. The mutant shows deficiency in glycolate oxidase activity and is non-viable in air; leaves become necrotic within 7 days and plants die within 15 days. When the mutant cells are shifted from high CO2 to air in light, they accumulate glycolate linearly for 6 hours to levels 7-fold higher than wild type and 11-fold higher after 25 hours. Belongs to the FMN-dependent alpha-hydroxy acid dehydrogenase family.