Catalytic electrochemistry of the bacterial Molybdoenzyme YcbX.

Molybdenum-dependent enzymes that can reduce N-hydroxylated substrates (e.g. N-hydroxyl-purines, amidoximes) are found in bacteria, plants and vertebrates. They are involved in the conversion of a wide range of N-hydroxylated organic compounds into their corresponding amines, and utilize various redox proteins (cytochrome b5, cyt b5 reductase, flavin reductase) to deliver reducing equivalents to the catalytic centre. Here we present catalytic electrochemistry of the bacterial enzyme YcbX from Escherichia coli utilizing the synthetic electron transfer mediator methyl viologen (MV2+). The electrochemically reduced form (MV+.) acts as an effective electron donor for YcbX. To immobilize YcbX on a glassy carbon electrode, a facile protein crosslinking approach was used with the crosslinker glutaraldehyde (GTA). The YcbX-modified electrode showed a catalytic response for the reduction of a broad range of N-hydroxylated substrates. The catalytic activity of YcbX was examined at different pH values exhibiting an optimum at pH 7.5 and a bell-shaped pH profile with deactivation through deprotonation (pKa1 9.1) or protonation (pKa2 6.1). Electrochemical simulation was employed to obtain new biochemical data for YcbX, in its reaction with methyl viologen and the organic substrates 6-N-hydroxylaminopurine (6-HAP) and benzamidoxime (BA).

Electrochemically driven catalysis of the bacterial molybdenum enzyme YiiM.

The Mo-dependent enzyme YiiM enzyme from Escherichia coli is a member of the sulfite oxidase family and shares many similarities with the well-studied human mitochondrial amidoxime reducing component (mARC). We have investigated YiiM catalysis using electrochemical and spectroscopic methods. EPR monitored redox potentiometry found the active site redox potentials to be MoVI/V -0.02 V and MoV/IV -0.12 V vs NHE at pH 7.2. In the presence of methyl viologen as an electrochemically reduced electron donor, YiiM catalysis was studied with a range of potential substrates. YiiM preferentially reduces N-hydroxylated compounds such as hydroxylamines, amidoximes, N-hydroxypurines and N-hydroxyureas but shows little or no activity against amine-oxides or sulfoxides. The pH optimum for catalysis was 7.1 and a bell-shaped pH profile was found with pKa values of 6.2 and 8.1 either side of this optimum that are associated with protonation/deprotonations that modulate activity. Simulation of the experimental voltammetry elucidated kinetic parameters associated with YiiM catalysis with the substrates 6-hydroxyaminopurine and benzamidoxime.