Abstract
The two-domain multicopper oxidases are proposed to be key intermediates in the evolution of three-domain multicopper oxidases. A number of two-domain multicopper oxidases have been identified from genome sequences and are classified as type A, type B, or type C on the basis of the predicted location of the type 1 copper center. The crystal structure of blue copper oxidase, a type C two-domain multicopper oxidase from Nitrosomonas europaea, has been determined to 1.9 A resolution. Blue copper oxidase is a trimer, of which each subunit comprises two cupredoxin domains. Each subunit houses a type 1 copper site in domain 1 and a type 2/type 3 trinuclear copper cluster at the subunit-subunit interface. The coordination geometry at the trinuclear copper site is consistent with reduction of the copper ions. Although the overall architecture of blue copper oxidase is similar to nitrite reductases, detailed structural alignments show that the fold and domain orientation more closely resemble the three-domain multicopper oxidases. These observations have important implications for the evolution of nitrite reductases and multicopper oxidases.
Highlights
Multicopper oxidases (MCOs)2 are a widely distributed class of enzymes with diverse functions ranging from copper and iron metabolism to polyphenol oxidation
MCOs contain four copper ions arranged in two sites: a blue type 1 mononuclear copper center (T1) and a trinuclear copper cluster (T2/T3) consisting of a normal type 2 copper center (T2) and dinuclear type 3 (T3) center [1,2,3]
Of structural similarities, MCOs are often grouped with copper nitrite reductases (NIRs), which contain both T1 and T2 sites [6], and are collectively referred to as multicopper blue proteins (MCBPs) [7]
Summary
Cell Growth—N. europaea cells were grown in batch cultures at 30 °C in the dark [20, 21]. Each large scale cell culture (closed high density polyethylene dome tanks, 120-liter cultures) was inoculated with the contents of a carboy. The concentrated cells were collected by centrifugation and resuspended with an equal volume of sodium phosphate buffer (50 mM NaH2PO4, 2 mM MgCl2). This resuspension was flash-frozen in liquid nitrogen and stored at Ϫ80 °C until used. BCO Purification and Activity—For protein isolation, N. europaea cells were sonicated and centrifuged for 30 min at 129,000 ϫ g. The final model (Table 1) consists of residues 44 –361 for each of the six chains (A–F), 24 copper ions, 2,009 water molecules, and four glycerol molecules. The BCO model used for pairwise superposition onto 3dMCOs consisted of chain A
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