Electron transfer and redox metalloenzyme catalysis at the single-molecule level

Voltammetry based on single-crystal, atomically-planar metal electrodes is novel in bioelectrochemistry. Together with in situ scanning tunneling microscopy (STM) directly in aqueous buffer, single-crystal voltammetry has disclosed new detail in molecular adsorption and interfacial electron transfer (ET). Image interpretation requires, however, theoretical support, as STM represents both electronic and topographic features. Molecules with accessible redox levels offer other insight into electron tunneling mechanisms, addressed in detail for ET metalloproteins. We present here in situ STM of the blue redox metalloenzyme copper nitrite reductase (Achromobacter xylosoxidans, AxCuNiR) on Au(111) electrode surfaces modified by a self-assembled cysteamine monolayer. AxCuNiR displays strong nitrite reduction waves in this environment. AxCuNiR/cysteamine/ Au(111) surfaces were imaged at KNO2 concentrations where most of the enzyme is in the enzyme-substrate bound state. Molecular resolution for both cysteamine/Au(111) and AxCuNiR/cysteamine/ Au(111) electrode surfaces was achieved. The enzyme coverage is about 1.5 x 10(-13) Mol cm(-2), which is low compared with an ideal close-packed monolayer. The adlayer behaves as an assembly of individual molecules, reflected in distributions of molecular appearance, although a number of molecules do show the triangular shape of the trimeric AxCuNiR structure. The apparent average molecular height is about 11 Angstrom. This suggests that details of electronic structures and larger assemblies are needed to disentangle enzyme mechanisms at the single-molecule level.