Figure 1.

Formation of a hydroxide/hydronium ion pair upon Cu_A reduction, modeled on bovine oxidase oxidized and reduced structures. A comparison of the oxidized (A) and reduced (B) structures of bovine oxidase indicates a change in the protonation status and bonding of two water molecules. (i) W1_E198^II. W1_E198^II is HOH2289 in the bovine 2DYR (Ox) and HOH2291 in the bovine 2EIJ (Red) structures. The distances indicate that W1_E198^II switches between a neutral water molecule and a hydronium ion upon reduction. This switch in protonation status is accompanied by its movement of 1.1 Å toward the free carboxylate oxygen of E198^II (OE2 in both structures). Upon Cu_A reduction H⁺−W1_E198^II becomes ionically bonded to the free carboxylate oxygen of E198^II, the bond distance shortening from 3.7 to 2.6 Å. (ii) W1_Mg. There are three waters bonded to the Mg ion, but only one can undergo deprotonation, W1_Mg (HOH2032). W2_Mg and W3_Mg (HOH2031, HOH2033) are pinned by the Mg(II) ion and the carboxylate D173^II (in the neutral state). A change in bond length between ⁽¹⁻⁾HO−W1_Mg/W1_Mg and the Mg(II) cation is not resolved in the bovine crystal structures; i.e., it is 2.3 Å in both structures. However, the bond lengths of W1_Mg to the two nearest water molecules, W1_H291 and W1_T127 (HOH2020 and HOH2054), do show a considerable shrinkage upon reduction, 2.9 to 2.8 Å and 3.3 to 2.9 Å, respectively, due to increased ionic bonding character as W1_Mg is converted to ⁽¹⁻⁾HO−W1_Mg. The bond angles and bond lengths of W1_Mg to W1_H291 and W1_T127 are shown in the insert of panels A and B.