Communication with the Mononuclear Molybdoenzymes: Emerging Opportunities and Applications in Redox Enzyme Biosensors

PAUL V. BERNHARDT

Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane 4072, Australia

1.1 Introduction – the Three Mo Enzyme Families

The mononuclear Mo-enzymes are remarkable in their coherence of active site structure and function yet equally interesting in the diversity of substrates that they are capable of oxidising or reducing. Apart from the well-studied enzyme nitrogenase, where the Mo ion is found within a S-bridged cluster of metals including Fe, all other enzymes containing Mo bear a single metal at the active site. A recent exception to this may be the novel Mo enzyme CO dehydrogenase, where a Cu ion shares a sulfide bridging ligand with the Mo ion at the active site.

All known enzymes from this family bear either one or two bidentate pterindithiolene (molybdopterin, MPT) ligands bound to the Mo ion at the active site (Figure 1.1). Hille proposed a classification of this group of enzymes into three families based on the coordination environment of the metal as shown in Figure 1.1.

At this time, enzymes from the DMSO reductase family (the most diverse of all) have only been found in bacteria and archea whilst enzymes from the other two families are found in all forms of life.

1.2 Mechanism

Although subtle differences exist in the mechanism of the mononuclear Mo enzymes, as a starting point, the reactions catalysed by this enzyme superfamily can be generalised by eqn (1) written in either the forward (reductase) or reverse (oxidase/dehydrogenase) direction. The substrates represented by the generic symbols Z and ZO are apparent from the names of the respective oxidases/ dehydrogenases (Z) or reductases (ZO) shown in Figure 1.1.

ZO + 2e- + 2H+ -> Z + H2O (1)

The use of Mo enzymes in electrochemically driven (amperometric) biosensors relies on connecting a working electrode with the redox active species involved in the catalytic cycle i.e. the enzyme and/or its substrates and products. The electrons required to sustain catalysis are provided or accepted by the electrode rather than the enzyme's natural cosubstrate. The various ways in which this can be done are summarised in the following section, but the most relevant point is that the Mo ion at the active site always cycles between its MoVI and MoIV oxidation states during catalysis and an O-donor ligand (oxo or hydroxo) is exchanged with the substrate during turnover. The MoVI form is the catalytically active form of the oxidases/dehydrogenases whilst the reductases must be reduced to MoIV before turnover can commence. The MoV form is a thermodynamically stable intermediate in most, but not all, cases, but it is incapable of turning over substrates in either direction. However, it may become an important rate-limiting intermediate in some cases.

Ligands bonded to the Mo ion are activated by coordination to perform some remarkable bond-breaking and formation reactions that otherwise do not occur in the absence of the enzyme. In the well-studied xanthine oxidor-eductases, a hydroxo ligand participates in a base-assisted nucleophilic attack at C-8 of xanthine coupled with a hydride abstraction by the sulfido ligand (Figure 1.2A). This mechanism is significantly di?erent from that seen in enzymes from the sulfite oxidase (Figure 1.2B) and DMSO reductase (Figure 1.2C) families where an oxo ligand is exchanged directly between the Mo ion and substrate during turnover.

1.3 Amperometric Biosensors

The development of enzyme-based biosensors in general has evolved over recent times as methods for addressing the active sites of enzymes have become better understood. Initially, enzyme electrochemistry relied upon the voltammetric detection of either the product or cosubstrate (so-called first-generation biosensors, Figure 1.3). The most common analyte that has been detected in this way is hydrogen peroxide, a typical product of oxidase enzyme turnover where the cosubstrate dioxygen is reduced in a two-electron proton-coupled reaction by the enzyme after substrate turnover. Alternatively the product itself may be electroactive but this is exceptional. A complementary approach is to monitor the depletion of cosubstrate, e.g. dioxygen, during turnover. However, this approach has limitations as variations in dioxygen concentrations may result from changes to the solution during analysis, e.g. temperature, stirring etc., and thus give false readings.

Second generation biosensors removed the natural cosubstrate from the system altogether, replacing it with a small molecule electron transfer mediator e.g. a redox active coordination compound or organic molecule. This approach has its roots in traditional enzyme solution assays where a mediator is oxidised or reduced chemically rather than electrochemically to drive the catalytic reaction. In electrochemistry, the mediator serves the dual purpose of (i) undergoing homogeneous electron transfer with the enzyme to restore it to its active form following turnover and (ii) undergoing heterogeneous electron transfer with the working electrode to provide the current that quantifies the enzyme-substrate reaction. This approach underpins most commercial enzyme biosensors to date including the glucose oxidase biosensor, which utilised a ferrocenium mediator (rather than dioxygen) as its artificial cosubstrate. The use of redox active polymers adsorbed on the electrode also comes under this classification. In all cases it should be emphasised that the currents observed are due to the mediator and they appear at the formal potential of the mediator and not of the enzyme. Ideally the redox potential of the mediator is in the vicinity of that of the active site. This avoids excessively large overpotentials which may lead to non-specific redox reactions with species in the sample other than the substrate.

The final approach (third generation) is to remove all cosubstrates from the system (natural or artificial) and to achieve direct electron exchange between the enzyme and the electrode. Although this has yet to...