Electron transfer ascorbate oxidase

While there is at present no full understanding as to why plastocyanin should require two sites for reaction, there is now much evidence detailing this two-site reactivity. Moreover, the recent X-ray crystal structure of ascorbate oxidase (which has 4 Cu atoms per molecule) has indicated a plastocyanin-like domain, with the two type 3 Cu s (in close proximity with the type 2 Cu) located at the remote site. Fig. 2 [5]. Since electrons are transferred, from the type 1 Cu to O2 bound at the type 3 center this structure defines two very similar through-bond routes for biological electron transfer. 

The recent X-ray crystal structure of ascorbate oxidase [6] has indicated the relative positions of type 1, 2 and 3 Cu centers. The type 1 center is in a plastocyanin like domain, and is the primary acceptor of electrons from substrate. The shortest pathway for electron transfer from the type 1 to type 3 Cu s is the bifurcated path via Cys508 and either His 507 or His509. The two histidines are part of the plastocyanin-like domain, and serve also to coordinate the type 3 Cu s, Fig. 2. The His507 to Cys508 bonding is similar to that of Tyr83... 

Figure 1 also contains a drawing of the electronic structure of plastocy-anin (Penfield et al., 1981, 1985), oriented according to the first part of Fig. 1. The d-orbital plane appears to be normal to the peptide plane that is extended by the hydrogen-bonding pair the it orbital of the thiolate would then also be affected, poising the cluster for electron transfer and, interestingly, apparently in the direction utilized by the multi-copper protein ascorbate oxidase (see Section V,A).

Copper has an essential role in a number of enzymes, notably those involved in the catalysis of electron transfer and in the transport of dioxygen and the catalysis of its reactions. The latter topic is discussed in Section 62.1.12. Hemocyanin, the copper-containing dioxygen carrier, is considered in Section 62.1.12.3.8, while the important role of copper in oxidases is exemplified in cytochrome oxidase, the terminal member of the mitochondrial electron-transfer chain (62.1.12.4), the multicopper blue oxidases such as laccase, ascorbate oxidase and ceruloplasmin (62.1.12.6) and the non-blue oxidases (62.12.7). Copper is also involved in the Cu/Zn-superoxide dismutases (62.1.12.8.1) and a number of hydroxylases, such as tyrosinase (62.1.12.11.2) and dopamine-jS-hydroxylase (62.1.12.11.3). Tyrosinase and hemocyanin have similar binuclear copper centres. 

The most common metal encountered in electron transfer systems is iron, although copper and manganese play vital functions. Merely to emphasise the complexity of the catalysts that are used in biology, the structures of the active sites of ascorbate oxidase (Fig. 10-11) and superoxide dismutase (Fig. 10-12) are presented. It is clear that we have only just begun to understand the exact ways in which metal ions are used to control the reactivity of small molecules in biological systems. 

Figure 8. Proposed electron transfer pathway in blue copper proteins. The plastocyanin wave function contours have been superimposed on the blue copper (type 1) site in ascorbate oxidase (40). The contour shows the substantial electron delocalization onto the cysteine Spir orbital that activates electron transfer to the trinuclear copper cluster at 12.5 A from the blue copper site. This low-energy, intense Cys Sp - Cu charge-transfer transition provides an effective hole superexchange mechanism for rapid long-range electron transfer between these sites (2, 3, 28).

Preliminary observation of additional electron density at this fourth coordination position of Cu-2 upon soaking crystals with N02 is consistent with this idea. Thus, from the structural data it would appear that Cu-1 is a type 1 center that functions to transfer electrons to the catalytic Cu-2 ion (See Note Added in Proof). It has been suggested, largely on the basis of electronic structural considerations (27, 28), that the Cys-136-His-135 link between Cu-1 and Cu-2 is a possible conduit for electron transfer between the two sites. An analogous dipeptide bridge between the type 1 center and the catalytic tricopper cluster in ascorbate oxidase (29, 30) may function similarly. Indeed, other close similarities between protein domains in ascorbate oxidase and NiR have been noted (17). 

Ascorbate oxidase is a tetramer each subunit has 552 amino acids and contains 4 copper ions, the type-I blue copper center and the adjacent trinuclear center (arranged as a type-n center and a type-in dinuclear center) separated by /S-sheets (Figure 20) °. Ascorbate is oxidized to dehydroascorbate by dioxygen however, it is not bound directly to the metal center to be oxidized, but is proposed to bind near the type-I Cu site which may facihtate electron transfer to oxygen, presumably in the tri-Cu cluster site. Since humans cannot synthesize ascorbic acid, conservation of this important compound is highly regulated. For example, the oxidized ascorbate can be transported into red blood... 

The driving force, calculated from the difference in the redox potentials ( + 344 mV for the type-1 copper in ascorbate oxidase (see Table VII) +295 mV for the couple ascorbate/ascorbate-free radical (176)) is 49 mV. In the proposed modeled encounter complex (74), there is a short distance of about 7 A between the two redox centers (distance GUI—01 ASC = 6.8 A distance GUI—02 ASG = 7.5 A) and an effective parallel arrangement of the rings, with good overlap of the TT-electron density systems facilitating a rapid electron transfer (see Fig. 15). 

The group of small plant proteins, azurin, stellacyanin, and plasto-cyanin, appear to be electron transfer proteins. They are listed because they share a type of copper site with the intensely blue representatives of the first class like laccase and ascorbate oxidase. The evidence that they participate in plant electron transfer chains remains circumstantial. Azurins, for example, purify along with well-known respiratory chain proteins like cytochrome C. A good deal of evidence exists, however, that plastocyanin is important in the photoreduction of NADP see below). 

Scheller et al. reported amperometric pyruvate sensors by potentiostatic co-pol5nmerization of Os(bipy)2pyridineCl-modified pyrrole monomer and thiophene on platinized glassy carbon electrodes on which pyruvate oxidase was adsorbed [78]. This pol5dhiophene based redox pol5uner was reported to have excellent electron transfer properties with significantly improved stability compared with polypyrrole as they are not affected by oxygen [79]. However, notable interference by ascorbate needs to be eliminated. 

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