Reexamining experimental data of single-molecule fluorescence correlation spectroscopy for cholesterol oxidase, we find that the existing Michaelis-Menten models with dynamical disorder cannot explain strong correlations between subsequent turnover cycles revealed in the diagonal feature in the joint statistical distribution of adjacent ''on'' times of this enzyme. We suggest that functional conformational motions representing ordered sequences of transitions between a set of conformational substates are involved, along with equilibrium conformational fluctuations in the turnover cycle of cholesterol oxidase. A two-channel model of singleenzyme dynamics, including a slow functional conformational motion in one of the channels, is proposed that allows us to reproduce such strong correlations.
Functionally important conformational motions are nonequilibrium processes that lead from one state of a protein to another and follow binding or dissociation of a ligand (1, 2). In enzymes, such ordered slow conformational motions can constitute an inherent part of a turnover cycle. Their functions may consist of transporting a substrate to the active center inside a macromolecule or bringing it into an appropriate arrangement with respect to this center. Generally, these are physical intramolecular motions that are needed for the occurrence of catalytic conversion and must precede it. Using time-resolved x-ray methods, functional conformational motions in Ha-ras p21 protein on GTP hydrolysis (3) and in a cytochrome P-450 enzyme (4) were recorded. In these experiments, enzyme molecules formed a crystal but could nonetheless perform their characteristic catalytic cycles. Today, many examples of ordered conformational motions in proteins, resolved by x-ray crystallography methods, are known (5-7) (see also the database at http:͞͞molmovdb.mbb.yale.edu͞molmovdb). Thermal conformational fluctuations for single macromolecules in solution were observed by using fluorescence correlation spectroscopy (FCS) (9, 10) and FRET methods (11). Conformational movements of enzymes in aqueous solvents could also be detected by NMR (12). Using FRET measurements, nonequilibrium conformational changes in single molecules of T4 lysozyme under reaction conditions were observed (13). Single-molecule FCS provides a powerful tool for monitoring chemical transitions during catalytic turnover cycles of individual molecules, and such experiments have been already performed for a number of enzymes, including cholesterol oxidase (14) and horseradish peroxidase (15,16). Simultaneous monitoring of chemical turnover cycles and physical processes of conformational changes was not, however, possible in the single-molecule experiments. Therefore, functional conformational motions during a turnover cycle for enzymic reactions in solution could not be identified. Nonetheless, the presence of such functional motions can be deduced by special statistical analysis of experimental data. In a previous publication, such statistical analysis, based on the memory functio...