Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

摘要

A single basic residue above the si-face of the flavin ring is thensite of oxygen activation in glucose oxidase (GOX) (His516) andnmonomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures ofnboth flavoenzymes exhibit a small pocket at the oxygen activation site thatnmight provide a preorganized binding site for superoxide anion, annobligatory intermediate in the two-electron reduction of oxygen. Chloridenbinds at these polar oxygen activation sites, as judged by solution andnstructural studies. First, chloride forms spectrally detectable complexesnwith GOX andMSOX. The protonated formofHis516 is required for tightnbinding of chloride to oxidized GOX and for rapid reaction of reducednGOX with oxygen. Formation of a binary MSOX 3nchloride complex requires Lys265 and is not observed with Lys265Met. Bindingnof chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavinnring. Definitive evidence is provided by crystal structures determined for a binary MSOX 3nchloride complex and a ternarynMSOX 3nchloride 3nMTA complex. Chloride binds in the small pocket at a position otherwise occupied by a watermolecule and formsnhydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two waternmolecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is anneffective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gasmethod that is usednto map nonpolar oxygen-binding cavities.