Abstract
Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.5-Å resolutions. These structures showed different copper site forms (met/deoxy and deoxy) and also differed from the copper site observed in the previously solved structure of AoCO4. We also analysed the electron density maps of all of the 56 CBC enzyme structures available in the protein data bank (PDB) and found that many of the published structures have vague copper sites. Some of the copper sites were then re-refined to find a better fit to the observed electron density. General problems in the refinement of metalloproteins and metal centres are discussed.
Highlights
Coupled binuclear copper (CBC) proteins contain a type-3 copper site that reversibly binds dioxygen [1]
The CBC protein family consists of oxygen carrier proteins haemocyanins, and various oxidative enzymes, including tyrosinases (EC 1.14.18.1), catechol oxidases (EC 1.10.3.1), and o-aminophenol oxidases (EC 1.10.3. 4) [2]
While the dioxygen binding capacity is a common feature of haemocyanins and enzymes in the CBC family, these enzymes often exhibit distinct substrate preferences for the catalysed oxidative reactions
Summary
Coupled binuclear copper (CBC) proteins contain a type-3 copper site that reversibly binds dioxygen [1]. The CBC protein family consists of oxygen carrier proteins haemocyanins, and various oxidative enzymes, including tyrosinases (EC 1.14.18.1), catechol oxidases (EC 1.10.3.1), and o-aminophenol oxidases While the dioxygen binding capacity is a common feature of haemocyanins and enzymes in the CBC family, these enzymes often exhibit distinct substrate preferences for the catalysed oxidative reactions. Catechol oxidase only catalyses the latter reaction (Fig 1), and o-aminophenol oxidase catalyses oxidation of o-aminophenols to o-quinoneimines in the grixazone biosynthetic pathway [3]. All enzymes that belong to the CBC family utilize molecular oxygen, which is the final electron acceptor and is reduced to water. In addition to the above-mentioned CBC enzymes, a novel
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