Competition between Photoinduced Electron Transfer and Resonance Energy Transfer in an Example of Substituted Cytochrome c–Quantum Dot Systems

Colloidal quantum dots (QDs) are nanoparticles that are able to photoreduce redox proteins by electron transfer (ET). QDs are also able to transfer energy by resonance energy transfer (RET). Here, we address the question of the competition between these two routes of QDs’ excitation quenching, using cadmium telluride QDs and cytochrome c (CytC) or its metal-substituted derivatives. We used both oxidized and reduced versions of native CytC, as well as fluorescent, nonreducible Zn(II)CytC, Sn(II)CytC, and metal-free porphyrin CytC. We found that all of the CytC versions quench QD fluorescence, although the interaction may be described differently in terms of static and dynamic quenching. QDs may be quenchers of fluorescent CytC derivatives, with significant differences in effectiveness depending on QD size. SnCytC and porphyrin CytC increased the rate of Fe(III)CytC photoreduction, and Fe(II)CytC slightly decreased the rate and ZnCytC presence significantly decreased the rate and final level of reduced FeCytC. These might be partially explained by the tendency to form a stable complex between protein and QDs, which promoted RET and collisional quenching. Our findings show that there is a net preference for photoinduced ET over other ways of energy transfer, at least partially, due to a lack of donors, regenerating a hole at QDs and leading to irreversibility of ET events. There may also be a common part of pathways leading to photoinduced ET and RET. The nature of synergistic action observed in some cases allows the hypothesis that RET may be an additional way to power up the ET.


Fluorescence data fitting
Fluorescence decays were fitted using OriginPro 2015 (OriginLab Corporation, USA) with a multicomponent exponential decay equation: where I is the recorded intensity, I0 is the intensity offset, t is the time of measurement with t0 time offset, Ai is the amplitude of the ith component, τi is the lifetime of the ith component and n is the total number of decay components. The satisfactory fits were obtained for n = 2 for fluorescent CytC derivatives and n = 3 for QDs.
The average fluorescence lifetimes (τav) were calculated using the amplitude-weighted mean: .
Plots of fluorescence intensity and lifetime as functions of the quencher concentration obtained from titration experiments were transformed to the Stern-Volmer F0/F and τav0/τav plots, where F0 and τav0 are fluorescence intensity and average lifetime in the absence of quencher and F and τavin the given concentration of quencher. The Stern-Volmer plots were used to calculate the values of quenching constants. In the case of equal slopes of F0/F and τav0/τav plots, the plots were fitted to the dynamic quenching equation: where [Q] is the molar quencher concentration and KSV is the dynamic (Stern-Volmer) quenching constant. When the value of F0/F slope was greater than τav0/τav line slope, KSV was calculated on the basis of τav0/τav plot. Then, F0/F plots were fitted to the equation for the combined (dynamic and static) mechanism of quenching, with a fixed value of KSV: where Ka is the static quenching constant, and n is the theoretical number of binding sites for the quencher on the fluorophore surface.

FCS measurements
Alexa Fluor 488 dye (A488; ThermoFisher) with a diffusion coefficient of 414 μm 2 s -1 at 25 °C in water 1 was used before the use of every cover slide for calibration of confocal volume after correction of the A488 diffusion coefficient for the temperature dependence of the water viscosity. For fitting the obtained fluorescence intensity data, the intensity autocorrelation function for isotropic three-dimensional diffusion, including intersystem crossing (triplet state of fluorophore), was used: where t is the lag time, fT is the fraction of fluorophores in the triplet state with lifetime τT, Np is the average number of fluorescent particles in the focal volume, τD is the diffusion time of investigated particles and s is the so-called structural parameter equal κ = z0 / w0 (where w0 and z0 are, respectively, the lateral and axial radii of the confocal volume × -2 ). Diffusion coefficients were calculated based on the diffusion times obtained from data fitting: and then the hydrodynamic radii r were determined using the Einstein-Stokes equation: where kB is the Boltzmann constant (1.38 × 10 -23 J/K), T is the absolute temperature during measurements and η is the water viscosity at temperature T. cpp (counts per particle) parameters were calculated based on the relationship: where νC is the rate of photon counts expressed in Hz, and G(0) is the value of the intensity correlation function in time t = 0.
The concentrations of QDs were 0.3 μM for QD510 and 0.1 μM for QD550 and QD750 in 25 mM HEPES at pH 7.5. The CytC proteins were added in a 1:1 to1:100 CytC:QD molar ratio.

Bio-layer interferometry assay
Binding parameters of QD510-CytC pairs obtained by bio-layer interferometry (BLI) experiments using the Octet (ForteBio) instrument. The assays were performed at 25 °C. Nickel-nitrilotriacetic acid (NTA) sensors were coated during the assay with His-tag-mCherry protein (500 sec of incubation, 1 μM in 25 mM Tris, 500 mM NaCl and 10% glycerol at pH 7.4), which exhibits high affinity to both nickel ions and QDs and was used as a linker to attach QD510 to the sensors. Next, QD510 was immobilised (500 sec, 2 μM in 25 mM Tris, 500 mM NaCl and 10% glycerol at pH 7.4) on the sensors. The association of CytC derivatives (1 μM) was monitored in 25 mM HEPES at pH 7.4, followed by dissociation in the same buffer (500 sec and 600 sec, respectively). The constants were obtained from fitting of binding curves to the 1:1 binding model after subtraction of the reference data (sensors without QD510 loading were used as a control for the unspecific binding of CytC proteins to the sensor surface).

Flash photolysis
The transient absorption system was designed as described earlier. 2 Briefly, pump pulses (355 nm, 8 ns FWHM, 0.5 Hz repetition rate, 1 mJ) were generated by a Q-switched Nd:YAG laser (Continuum Surelite II). As the probe, a 150-W xenon arc lamp (Applied Photophysics, UK) was used either in pulsed (for time window <500 µs) or steady-current mode (for time window >500 μs). The probe passed through the sample with a 1 Hz repetition rate obtained using a shutter (Uniblitz). A monochromator (Acton Research Spectra Pro 300i) was used to disperse the probe light, and a photomultiplier (R928 Hamamatsu) coupled with a digital oscilloscope (Tektronix TDS 680 C) was used for detection. Samples (1 ml) were placed in a quartz cuvette (10 mm x 10 mm cross section). Measurements were performed at room temperature in 25 mM HEPES at pH 7.4 and deoxygenated with argon for at least 15 min. The concentrations of samples were adjusted to yield at least 0.4 absorbance at the excitation wavelength (355 nm), and they were 1 µM QD630, 25 μM Fe(III)CytC, 15 µM ZnCytC and 0.5 μM QD630 + 5 μM Fe(III)CytC when these two were mixed.       Two abnormalities from Stern-Volmer theory should be noted here. First is upward curvature of some τav0/τav plots (for titrations of QD510 by Fe(III)CytC, Fe(II)CytC and ZnCytC) what is in contradiction to standard assumption of the linearity of these plots. The possible explanation of this bimodal curve behavior is the presence of at least two processes decreasing the lifetime of QDs. For KSV derivation the tangent to initial τav0/τav plot was taken for fitting to dynamic quenching equation. Second anomaly is the slope value of τav0/τav curve for QD750 titration by ZnCytC exceeding ca. two times the slope value for F0/F plot. For this case, pure dynamic quenching was assigned and F0/F plot was taken for fitting and KSV calculation.

S 11
The temperature dependence of QD510 quenching by CytC derivatives Dynamic quenching is usually the result of transient interactions driven by thermal collisions of fluorophore and quencher particles. Hence, dynamic quenching efficiency increases in a temperaturedependent manner. The opposite is true for a static quenching: increasing temperature disturbs a molecular complex formation. The character of quenching, caused by CytC proteins on QD510 fluorescence, was also evaluated in the 10-40 C range of temperature (Fig. S7). Quenching efficiency of Fe(III)CytC, defined as F0/F ratio, decreases in higher temperatures, revealing a predominant static component. The effectiveness of QD510 quenching by ZnCytC and SnCytC increases with growing temperature, which indicates the significant dynamic component in their quenching mechanism. This is highlighted especially by the increasing τav0/τav ratiothe most potent dynamic quencher, ZnCytC, exhibits the largest temperature-dependent increase of this parameter.

S 16
Simulated quenching constant, representing the constant expected for no interaction between ET and RET, was constructed by taking the average values of points of titration curves for two given CytC forms (creating the theoretical titration curves lying equidistantly between two titration curves for given single CytC forms). Such a method was based on the assumption that the mixture, containing two ideally non-interacting assays, may be virtually represented by two mixtures containing each assay separately. Then, if two cuvettes containing each of the assays were placed simultaneously in the light pathway, the recorded signals would serve (after correction for concentration) as titration curves. Such a measurement is easy to carry out in a transmission or absorption mode. However, due to the specific geometry of a fluorometric apparatus, it is impossible to ensure equal illumination of joined cuvettes, and the necessary values are obtained by mathematical transformation from two independent measurements. The simulated data were then fitted to Stern-Volmer equations to calculate quenching constants.

Fig. S12
Procedure for calculation of quenching constants based on titration results. Depending on the character of CytC quencher, KSV value was obtained as a slope of F0/F plot (for dynamic quenchers) or τav0/τav plot (for combined mechanism of quenching). Ka value for static component of quenching was calculated by fitting of F0/F plot to Stern-Volmer equation for combined quenching mechanism (KSV value obtained from τav0/τav slope was fixed the during the fitting).

Fig. S17
Binding parameters of QD510-CytC pairs obtained by BLI experiments. QD510 was immobilized on Ni-NTA sensors (with His-tag-mCherry protein as linker) and binding of different CytC derivatives was measured. As control of unspecific binding sensors without QD510 immobilized were used. The binding constants were obtained from fitting of binding curves to the 1:1 binding model. The error bars represent standard errors of fitting of single measurement.