Memory landscapes of single-enzyme molecules

Abstract

Immobilized single horseradish peroxidase enzymes were observed by confocal fluorescence spectroscopy during catalysis of the oxidation reaction of the nonfluorescent dihydrorhodamine 6G substrate into the highly fluorescent product rhodamine 6G. By extracting only the non-Markovian behavior of the spectroscopic two-state process of enzyme-product complex formation and release, memory landscapes were generated for single-enzyme molecules. The memory landscapes can be used to discriminate between different origins of stretched exponential kinetics that are found in the first-order correlation analysis. Memory landscapes of single-enzyme data shows oscillations that are expected in a single-enzyme system that possesses a set of transient states. Alternative origins of the oscillations may not, however, be ruled out. The data and analysis indicate that substrate interaction with the enzyme selects a set of conformational substates for which the enzyme is active. Non-ergodic properties of a process of a molecule (12, 13) as well as non-exponential state transition probabilities for a single process (5) are both indicators of a complex behavior. Enlarged dynamic models on the single molecule level are then required as compared with models derived from standard chemical kinetics of an ensemble of molecules.Catalysis of the oxidation of the dihydrorhodamine 6G into rhodamine 6G by the enzyme horseradish peroxidase on the single enzyme level has recently been observed at room temperature (5). Horseradish peroxidase is a 44-kDa heme protein (14,15) and is an effective catalyst of the decomposition of hydrogen peroxide (H 2 O 2 ) in the presence of hydrogen donors (14, 15). The reaction is monitored by existing experimental methods (8) based on confocal fluorescence spectroscopy (16,17). We used the nonfluorescent substrate dihydrorhodamine 6G, which after oxidation yields the highly fluorescing rhodamine 6G fluorophore. Hence, direct observation of successive single substrate turnovers into product is made possible by means of fluorescence microscopy if a single enzyme molecule is observed. The enzyme, the substrate, and the enzyme-substrate complex are nonfluorescent. However, the enzyme-product complex (EP) (18) is fluorescent and is formed as the result of the substrate being oxidized while still bound to the enzyme; the enzyme-substrate complex transforms into a fluorescent EP. For each catalytic cycle, a new substrate is bound to the enzyme and is turned over into a product (EP), after which the product dissociates from the enzyme. Then, another substrate attaches to the enzyme, is turned over into a product (EP), and so on. The average binding time of the product (lifetime of EP) was determined in ref. 5 to be approximately 50 ms. The observable state from a spectroscopic viewpoint is the EP. All other states of the enzyme are nonfluorescent. It is assumed that the spectroscopic properties of the EP are unaffected by the oxidation state of the enzyme (e.g., 4 ϩ or 5 ϩ oxidized state) because we directly monitor the...

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…The activity of horseradish peroxidase (HRP) was investigated by Rigler and collaborators using FCS on immobilized molecules. 198,199 Oxidation of the nonfluorescent dye dihydrorhodamine 6G by HRP in the presence of H 2 O 2 leads to two fluorescent Rhodamine 6G molecules per enzyme cycle, each oxidation cycle consisting in the binding of the substrate, oxidation, and product release: (23) where the asterisk indicates the only visible species (free products are released and diffuse away). For all practical purposes, the authors modeled the system as a two-state model, with one invisible species (E) and one visible one (EP), interconverting as: (24) with effective rates k 1 and k −1 .…”

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…Different members of a seemingly homogeneous collection of molecules may exhibit different binding kinetics (so-called static disorder), or individual members of a population may exhibit binding kinetics that change over time (dynamic disorder) [11][12][13][14][15][16]. Characterizing these different phenomena and correlating them with information on structural conformation will increase our understanding of the relationship between activity and structure, an important factor in both binding and enzymatic activity [14,[17][18][19][20][21][22].…”

Michaelis-Menten reaction scheme as a unified approach towards the optimal restart problem

“…Non-exponential time distributions are, however, quite common in a variety of complex systems [36][37][38][39][40], and it has recently been recognized that enzymes are no exception in that regard [23,32,41,42]. This result is perhaps not surprising as catalysis is intrinsically coupled to the enzyme's internal degrees of freedom via a complex energy landscape [43] which can give rise to strong deviations from exponentiality and other anomalies [44][45][46][47][48]. Renewal theory can then be invoked to provide a generalized mathematical treatment of the MMRS [26].…”