Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

Abstract

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.ABEL trap | enzymology | electron transfer | fluorescence | catalysis M easurements made on the activity of single enzymes have revolutionized the picture of these molecular machines' mode of operation by providing evidence for static and dynamic heterogeneity, enabling the observation of transient intermediates, and allowing determination of the microscopic rate constants comprising complex catalytic cycles under turnover conditions (1-4). However, conclusions regarding enzyme dynamics require the observation of multiple turnovers of a single molecule with a requisite observation window of several seconds or more. Because this window is several orders of magnitude longer than the typical dwell time of a solution-phase molecule diffusing through a confocal detection volume, molecules that naturally exist in solution are often immobilized on a solid support (1-3, 5). Although gross comparison between properties measured on immobilized and freely diffusing biomolecules tends to show good agreement (1, 3), more subtle aspects of biomolecule behavior such as property distribution widths and shapes (6, 7) and conformational dynamics (6, 8) have been shown to be affected by immobilization, and in some cases, immobilization has been suspected of contributing to observed enzyme behavioral heterogeneity (4). In this report we take an important step toward viewing the richness of single-enzyme dynamics without immobilization by making observations of multiple turnover events of single solution-phase molecules of a blue nitrite reductase (bNiR), an important redox-active enzyme in bacterial denitrification (9).Prolonged solution-phase measurements are enabled through use of a specialized microfluidic trapping device that cancels the Brownian motion of a single emissive solution-phase object through the use of directed electroosmotic forces, the anti-Brownian electrokinetic (ABEL) trap (10, 11). The ABEL trap estimates particle position via a revolving laser spot phase-locked in a homebuilt analogue circuit to a restoring force gated by photon detection and applied via two orthogonal pairs of electrodes (11). This scheme results in the confinement of the object in a spatially homogeneous excitation volume for multiple seconds without immobilization and can be applied to single biomolecules substantially smaller than the large...

Copper Active Sites in Biology

“…Whether these two steps are ordered or if a random-sequential mechanism operates has been a matter of long debate in the literature. However, both a recent single-molecule study 1268 and a steady-state kinetics study considering the substrate dependence of turnover support a random-sequential mechanism, 1269 where either ET or substrate binding could occur as the first step (Figure 231, pathways A and B) and this view has become generally accepted. When both nitrite binding and T1 to T2 ET have occurred, the mechanism converges at a T2 reduced nitrite bound intermediate.…”

Single-molecule spectroscopy and imaging over the decades

“…10d). As another example 196 , an engineered FRET redox sensor was used to directly visualize the transitions between discrete redox states of the enzyme nitrite reductase (Fig. 10b), providing new insights into the mechanism of catalysis.…”

“…Adapted with permission from Ref. 196 (c) Counting the number of ADP molecules on a multi-subunit chaperonin enzyme (TRiC). Digital steps represent photobleaching of the individual Cy3-ADP molecules bound to the protein.…”

Characterization of a Nitrite Reductase Involved in Nitrifier Denitrification

“…This mechanism is often referred to as the "ordered" or the "bind first mechanism" (56). However, there is strong kinetic evidence as well as single molecule data indicating that CuNIRs may follow a "random sequential mechanism" in which either nitrite can bind first or electron transfer from the T1 to the T2 copper center can occur prior to nitrite binding (57,58). In the random sequential mechanism, the order of events is proposed to be dictated by substrate concentrations and pH.…”