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Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow. for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism. [ABSTRACT FROM AUTHOR]

The hydrophobic patch of azurin (AZ) from Pseudomonas aeruginosa is an important recognition surface for electron transfer (ET) reactions. The influence of changing the size of this region, by mutating the C- terminal copper-binding ioop, on the ET reactivity of AZ adsorbed on gold electrodes modified with alkanethiol self- assembled monolayers (SAMs) has been studied. The distance-dependence of ET kinetics measured by cyclic voltammetry using SAMs of variable chain length, demonstrates that the activation barrier for short-range ET is dominated by the dynamics of molecular rearrange- ments accompanying ET at the AZ-SAM interface. These include internal electric field-dependent low-amplitude protein motions and the reorganization of interfacial water molecules, but not protein reorientation. Interfacial molecular dynamics also control the kinetics of short- range ET for electrostatically and covalently immobilized cytochrome c. This mechanism therefore may be utilized for short-distance ET irrespective of the type of metal center, the surface electrostatic potential, and the nature of the protein∔SAM interaction. [ABSTRACT FROM AUTHOR]