Cobalt substitution supports an inner-sphere electron transfer mechanism for oxygen reduction in pea seedling amine oxidase

Abstract

Copper amine oxidases (CAOs) are a large family of proteins that use molecular oxygen to oxidize amines to aldehydes with the concomitant production of hydrogen peroxide and ammonia. CAOs utilize two cofactors for this reaction: topaquinone (TPQ) and a Cu(II) ion. Two mechanisms for oxygen reduction have been proposed for these enzymes. In one mechanism (involving inner-sphere electron transfer to O(2)), Cu(II) is reduced by TPQ, forming Cu(I), to which O(2) binds, forming a copper-superoxide complex. In an alternative mechanism (involving outer-sphere electron transfer to O(2)), O(2) is directly reduced by TPQ, without reduction of Cu(II). Substitution of Cu(II) with Co(II) has been used to distinguish between the two mechanisms in several CAOs. Because it is unlikely that Co(II) could be reduced to Co(I) in this environment, an inner-sphere mechanism, as described above, is prevented. We adapted metal replacement methods used for other CAOs to the amine oxidase from pea seedlings (PSAO). Cobalt-substituted PSAO (CoPSAO) displayed nominal catalytic activity: k(cat) is 4.7% of the native k(cat), and K(M) (O(2)) for CoPSAO is substantially (22-fold) higher. The greatly reduced turnover number for CoPSAO suggests that PSAO uses the inner-sphere mechanism, as has been predicted from (18)O isotope effect studies (Mukherjee et al. in J Am Chem Soc 130:9459-9473, 2008), and is catalytically compromised when constrained to operate via outer-sphere electron transfer to O(2). This study, together with previous work, provides strong evidence that CAOs use both proposed mechanisms, but each homolog may prefer one mechanism over the other.

Plant Copper Metalloenzymes As Prospects for New Metabolism Involving Aromatic Compounds

“…Lastly, the release of ammonia in the presence of water reoxidizes TPQ (Mills et al, 2012). The oxidative half-reaction uses indirect, inner-sphere electron transfer, and is demonstrated in pea seedling CuAO (Mills et al, 2012(Mills et al, , 2019.…”

“…The superoxide attacks the semiquinone of the TPQ ring, and proton-coupled electron transfer results in the iminoquinone form of TPQ with release of hydrogen peroxide ( Dooley et al, 2017 ). Lastly, the release of ammonia in the presence of water re-oxidizes TPQ ( Mills et al, 2012 ). The oxidative half-reaction uses indirect, inner-sphere electron transfer, and is demonstrated in pea seedling CuAO ( Mills et al, 2012 , 2019 ).…”

“…Lastly, the release of ammonia in the presence of water re-oxidizes TPQ ( Mills et al, 2012 ). The oxidative half-reaction uses indirect, inner-sphere electron transfer, and is demonstrated in pea seedling CuAO ( Mills et al, 2012 , 2019 ). The outer-sphere electron transfer mechanism, where reduced TPQ transfers an electron to dioxygen directly so the oxidization state of Cu(II) is constant, is used in fungi ( Mills et al, 2019 ).…”

The Role of Protein Crystallography in Defining the Mechanisms of Biogenesis and Catalysis in Copper Amine Oxidase

“…The issue of the first electron transfer from cofactor to O 2 is being actively pursued, and it has been suggested that CAOs may be capable of using more than one mechanism to reduce O 2 but that each different CAO has a clear preference for either inner or outer sphere electron transfer [89,103,104]. Regardless, O 2 ultimately accepts two electrons and two protons from the cofactor, yielding H 2 O 2 and the iminoquinone (G in Scheme 4).…”

The precursor form ofHansenula polymorphacopper amine oxidase 1 in complex with CuIand CoII

“…In contrast to the four-coordinate tetrahedrally bound cobalt ion in Co II -apoAGAO, Co II -AGAO contains six-coordinate cobalt ligated by the three histidine residues, axial and equatorial water ligands common to the native structure and an additional water molecule in an octahedral geometry. Such geometric differences may explain the inefficiency of Co II -PSAO in catalyzing substrate oxidation in relation to native Cu II -PSAO (Mills et al, 2012). It appears that differences in metal-coordination geometry, in conjunction with additional factors such as Lewis acidity and redox potential, coordinate to determine whether a metal can initiate biogenesis as well as sustain efficient catalysis in the mature CAO.…”