¹ Institute of Biomedical Chemistry, Moscow, Russia

² Foundation of Perspective Technologies and Novations, Moscow, Russia

³ Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus, Minsk, Belarus

Herein, we report the use of a nanotechnology-based approach for the study of enzyme-functionalized mica surfaces. Atomic force microscopy (AFM) has been employed for the determination of the catalytic activity of single molecules of heme-containing cytochrome P450 CYP102A1 (CYP102A1) enzyme, which was immobilized on the surface of a mica chip. Height fluctuations in individual molecules of the enzyme were measured under near-native conditions by AFM measurements in liquid using a cantilever with a 10 to 20 nm tip curvature radius. We have found that in the process of enzymatic catalysis, the mean amplitude of height fluctuations in individual enzyme molecules is 1.4-fold higher than that of enzyme molecules in an inactive state. The temperature dependence of the mean amplitude of height fluctuations in cytochrome CYP102A1 has been revealed, and the maximum of this dependence has been observed at 22 °C. The proposed nanotechnology-based approach can be employed in studies of a wide variety of enzymes, which are important for the development of novel diagnostic tests and systems for pharmaceutical analysis. The approach developed in our work will favor further miniaturization of enzyme-based biosensors and the transition from traditional sensors to nanobiosensors.

Keywords: cytochrome P450; atomic force microscopy; single-molecule enzymology; enzymatic activity; biosensor

In recent decades, the studies of single molecules of enzymes have attracted growing attention. At the same time, enzyme-based biosensor systems have found numerous applications in drug discovery [1] and in diagnostics [2]. Single-molecule studies of enzyme’s properties, including their enzymatic activity, have formed the very base of a new direction in biochemistry. This direction was called single-molecule enzymology [3]. In this connection, nanotechnology-based methods—atomic force microscopy (AFM) [4], nanowire-based [5], and nanopore-based [6,7] ones—are of use. The use of nanotechnology-based approaches to enzyme investigation favors further miniaturization of enzyme-based analytical devices, switching from traditional sensors to nanobiosensors. Nanowire- and nanoribbon-based biosensors, though providing very high detection sensitivity [5,8], do not currently allow the investigation of enzymatic reactions. On the contrary, nanopore-based sensing devices allow one to directly monitor the functioning of single-enzyme molecules, but require high concentrations of the enzyme of interest in the test solution [7].

Atomic force microscopy (AFM) is currently one of the most widely used methods of visualization and determination of the physicochemical properties of single molecules of proteins [9], including enzymes [4], nucleic acids [10], their macromolecular complexes [11,12,13], and viral particles [14] immobilized on an atomically smooth surface of a solid substrate [15,16,17,18]. For the visualization of proteins and enzymes on such a surface, one of the dynamic modes of AFM—semi-contact mode (tapping mode)—is commonly employed [15]. This allows one to minimize the impact of the AFM probe on the studied macromolecules in the process of their visualization in order to avoid protein destruction [19]. These features of AFM allow its use for the visualization of monomers and oligomers of proteins and for their identification by the height of their AFM images, as was demonstrated with the examples of putidaredoxin reductase (PdR) and cytochrome P450 102A1 [16,20]. AFM also allows one to distinguish between single protein molecules and complexes with their redox partners in complex multicomponent systems, as was demonstrated with examples of cytochrome P450-containing monooxygenase systems [15,17,21,22]. In contrast to nanopore-based sensing devices, AFM-based techniques allow one to detect the molecules of interest even at ultra-low concentrations [23].

It should be emphasized that in the present work, the tapping mode of AFM has been employed for not only the visualization, but also for the determination of the enzymatic activity of CYP102A1 enzyme, which pertains to the superfamily of heme-containing cytochromes P450. Cytochromes P450 plays a key role in xenobiotic metabolism [24,25]. Since these enzymes are key actors in drug metabolism [25], they represent important components of analytical systems for studying drug–drug interactions [1].

CYP102A1 catalyzes the monooxygenation of fatty acids [26]. CYP102A1 is a self-sufficient enzyme, and this means that no partner protein is required for its functioning [26,27]. This makes CYP102A1 a very convenient model enzyme for experimental studies of cytochromes P450, and this is why it has been selected as an object in our study, allowing us to simplify the scheme of experiment on the AFM-based determination of the cytochrome’s enzymatic activity. In solution, CYP102A1 is known to exist in the form of monomers, dimers, and higher-order oligomers [20,28,29]. Neeli et al. [29] reported the Kd value for the dimerization of CYP102A1 monomers to be 1.1 ± 0.2 nM. In a number of studies [29,30,31,32,33], the enzymatic activity of CYP102A1 in the reaction of lauric acid hydroxylation (in (ω-1), (ω-2), and (ω-3) positions [34]) in solution was investigated; the turnover rate (kcat) values reported in these studies differ significantly: kcat = 53 s−1 [29], 46 s−1 [30], 86 s−1 [31], 26 s−1 [32], and 2 s−1 [33].

Our study is aimed at the elaboration and enhancement of the AFM-based method of determination of individual molecules of cytochromes pertaining to the P450 superfamily. In the experiments reported herein, we have measured the enzymatic activity of CYP102A1 by AFM, directly monitoring the behavior of individual molecules of the enzyme. This is in contrast to the approach employed in the above-mentioned studies [29,30,31,32,33], in which enzymatic activity values were determined by spectroscopic or isotopic (radioactive labeling) methods. In the latter method, the signal is received from a large ensemble of enzyme molecules, and the activity value is determined in such a way that it is, thus, averaged over this molecular ensemble. In this respect, the AFM-based determination of the activity of individual molecules is fundamentally different.

The AFM-based determination of CYP102A1 enzymatic activity is based on the monitoring of an increase in the amplitude of the enzyme molecules’ height fluctuations in the process of lauric acid hydroxylation. This AFM-based monitoring of CYP102A1 height value throughout the catalytic process has allowed us to register a 1.4-fold increase in the mean amplitude of CYP102A1 height fluctuations upon the enzyme functioning. The catalytic cycle time is the mean value of time between events of maximum excitation of the CYP102A1 globule throughout its functioning during the observation period. The temperature dependence of CYP102A1 globule excitation in the process of enzyme functioning has been determined.

The nanotechnology-based approach proposed herein can be of use in the development of novel enzyme-based nanoscale biosensors. These enzyme-based biosensors can find their application in diagnostics and in drug discovery.

A total of 10 mM phosphate-buffered saline (Dulbecco’s modified; PBSD) with a pH of 7.4, containing 8 mM sodium phosphate, 2 mM potassium phosphate, 140 mM sodium chloride, and 10 mM potassium chloride was purchased from Pierce (USA). The sodium salt of lauric acid, NADPH, and 1-phenylimidazole were purchased from Sigma (St. Louis, MO, USA). Hydroxylauric acid standard (of 99.0% purity) was purchased from SynFine Research, Inc. (Richmond Hill, ON, Canada). All the aqueous solutions were prepared with ultrapure deionized water, which was purified with a Simplicity UV system (Millipore, Molsheim, France).

Wild-type cytochrome CYP102A1 was kindly donated by Professor Dr. A.W. Munro; the enzyme was expressed and prepared as described elsewhere [29]. The enzyme was also expressed by Dr. A.V. Grudo. The absorbance spectra of CYP102A1 were obtained with an Agilent Model 8453 diode array spectrophotometer (Agilent Deutschland GmbH, Waldbronn, Germany) at 25 °C. The concentration of purified CYP102A1 was determined according to the technique described by Omura and Sato [34] based on the differential absorbance spectrum of the carboxy complex of the enzyme’s reduced form, given that the extinction coefficient for the difference in absorbance at 450 and 490 nm was equal to 91 mM−1cm−1.

CYP102A1 enzyme was non-covalently immobilized onto freshly cleaved mica substrates by direct surface adsorption (SPI, West Chester, PA, USA). With this purpose, a 2 µL volume of 0.5 µM CYP102A1 solution in 10 mM PBSD buffer was dispensed onto a piece of freshly cleaved mica (SPI, USA). At that, the temperature of the enzyme solution was 4 to 10 °C. The enzyme solution was incubated on mica for three minutes and then washed away with deionized water. The so-treated substrate was placed into an AFM liquid cell filled with pure 2.5 mM PBSD. Such a low salt concentration in the buffer was used in order to avoid the influence of salt deposits on the substrate surface on the quality of AFM images [35].

In order to determine whether or not there is adsorption of non-specific objects on mica, the control blank experiments were also performed at the enzyme immobilization step. Namely, a 2 µL volume of 10 mM protein-free PBSD buffer was dispensed onto control mica, and incubated thereon instead of CYP102A1 enzyme solution for three minutes.

The AFM measurements were performed in the tapping mode in liquid with a Dimension 3100 atomic force microscope (Bruker, Billerica, MA, USA) equipped with DNP-S10 AFM probes (Bruker, USA; force constant 0.32 to 0.58 N/m; nominal curvature radius 10 to 20 nm). The purity of all the solutions was checked by AFM prior to their use in the AFM experiments; the height of non-specific objects revealed on mica during these checks did not exceed 0.5 nm. The temperature in the AFM liquid cell was kept constant and set to 22 °C—except for experiments on the determination of the temperature dependence of enzymatic activity. In the latter case, the temperature in the cell varied between 17 and 28 °C.

The AFM-based monitoring of enzymatic activity consisted of obtaining time dependence of the height fluctuations in individual molecules of CYP102A1 in the process of the catalytic cycle of lauric acid hydroxylation reaction according to the method described elsewhere [36]. With this purpose, a preliminary scanning of the substrate surface was performed in order to select an area containing the adsorbed enzyme molecule of interest. After that, the scanning of this selected area was started. At the moment when the scanning probe reached the middle region of the selected molecule, scanning in the slow scan direction (along the Y-axis) was disabled. In this way, a cross-section image of the selected enzyme molecule of interest was obtained in a time-base sweep. The scanning frequency was set to the highest value technically possible for this atomic force microscope (1 to 1.3 Hz).

The AFM-based monitoring of the enzymatic activity was performed in four sequential steps. Firstly, the measurements were performed in pure 2.5 mM PBSD with pH 7.4 (step 1). Secondly, sodium laurate (substrate) solution in buffer was added into the liquid cell to the final substrate concentration of 500 µM (step 2). Thirdly, the buffered solution of NADPH electron donor was added into the cell to its final concentration of 200 µM (step 3). In the fourth step, the catalytic reaction was stopped by the addition of 1-phenylimidazole inhibitor to its final concentration in the cell of 5 mM.

At all four steps, cross-section images of no less than ten different molecules on the substrate were obtained as described above. Observations upon the addition of the substrate, the electron donor, or the inhibitor were performed with a delay from the moment of addition of either of the enzymatic reaction components tlag~5 min. This time was required to stabilize the AFM scanning conditions after the hydrodynamic disturbance in the liquid in the cell, which occurred upon the addition of the solution with either of the reaction components.

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Ivanov, Y.D.; Bukharina, N.S.; Shumov, I.D.; Afonin, O.N.; Tatur, V.Y.; Grudo, A.V.; Archakov, A.I. AFM-Based Monitoring of Enzymatic Activity of Individual Molecules of Cytochrome CYP102A1. Biosensors 2025, 15 (5), 303. https://doi.org/10.3390/bios15050303