Purification and Properties of Cyclopentanone Oxygenase of Pseudomonas NCIB 9872

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Abstract
1 Cyclopentanone oxygenase from Pseudomonas NCIB 9872 has been purified some 40-fold. It gives a single peak in the ultracentrifuge and a single major protein band on polyacrylamide gels contaminated with about 5% of a slower migrating impurity. Flavin dissociates from the protein during electrophoresis. 2 The enzyme has a molecular weight of about 200000 and is a homopolymeric assemblage of either three or four subunits of molecular weight 54000–58000. 3 The prosthetic group is FAD and values of about 2.5 are typically obtained for the number of moles bound to each mole of holoenzyme. Some FAD probably dissociates during purification and it seems likely that each subunit binds one FAD in the undamaged protein. 4 The unitary stoichiometry of cyclopentanone, oxygen and NADPH is typical of mixedfunction oxygenases with external electron donors. The oxygenated product has been identified as 1-oxa-2-oxocyclohexane (5-valerolactone) and enzyme is therefore systematically named as cyclopentanone, NADPH: oxygen oxidoreductase (1,2-lactonizing) (EC 1.14.13.–). 5 Catalytically functional sulfhydryl groups are probably present but the biphasic nature of the inhibition curve when enzyme is titrated with p-hydroxymercuribenzoate reveals the presence of six titratable groups that are not all equivalent. 6 The enzyme has a pH optimum of 7.7. The Km for cyclopentanone is below 1 μM but not experimentally determinable. The response to variation of NADPH concentration is sigmoidal, indicative of homotrophic binding, a Hill plot gave an interaction number of 3, perhaps indicative of cooperativity between three subunits. 7 Anaerobic reduction of enzyme-bound FAD by addition of NADPH required only slightly in excess of two electron equivalents. No evidence for the formation of stable semiquinones was obtained in this or in photoreduction experiments. 8 Though the enzyme displays sensitivity towards transition metal chelating agents the small amounts of Fe and Cu present make a role in electron transport unlikely. The possibility that they may be involved in maintenance of quaternary structure has not been investigated.