Flavin-Linked Erv-Family Sulfhydryl Oxidases Release Superoxide Anion during Catalytic Turnover.

Typically, simple fiavoprotein oxidases couple the oxidation of their substrates with the formation of hydrogen per-oxide without release of significant levels of the superonde ion. However, two evolutionarily related single-domain sulihydryl oxidases (Erv2p; a yeast endoplasmic reticulum resident protein and augmenter of liver regeneration, ALR, an enzyme predom-inantly found in the mitochondrial intermembrane) release up to ∼30% of the oxygen they reduce as the superoxide ion. Both enzymes oxidize dithiol substrates via a redox-active disulfide adjacent to the flavin cofzctor within the helix-rich Erv domain. Subsequent reduction of the fiavin is followed by transfer of reduc-ing equivalents to molecular oxygen. Superoxide release was initially detected using tris(3-hydroxypropyl)phosphine (TI-TP) as an alter-native reducing substrate to dithiothreitol (DTT). Till', and other phosphines, showed anomalously high turnover numbers with Erv2p and ALR in the oxygen electrode, but oxygen consumption was drastically suppressed upon the addition of superoxide dismutase. The superoxide ion initiates a radical chain reaction promoting the aerobic oxidation of phosphines with the fonnation of hydrogen peroxide. Use of a known flux of superoxide generated by the xanthine/xanthine oxidase system showed that one superoxide ion stimulates the reduction of 27 and 4.5 molecules of oxygen using THP and tris(2-carboxyethyl)phosphine (TCEP), respectively. This superoxide-dependent ampli-fication of oxygen consumption by phosphines provides a new kinetic method for the detection of superoxide. Superoxide release was also observed by a standard chemilusninescence method using a luciferin analogue (MCLA) when 2 mM DTI' was employed as a substrate of Erv2p and ALR. The percentage of superoxide released from Erv2p increased to ~-~6S% when mono-meric mutants of the normally homodimeric enzyme were used. In contrast~ monomeric multidomain quiescin sulihydryl oxidase enzymes that also contain an Erv FAD-binding fold release only 1-5% of their total reduced oxygen species as the superoxide ion. Aspects of the mechanism and possible physiological significance of superoxide release from these Erv-domain flavoproteins are discussed. [ABSTRACT FROM AUTHOR]