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Abstract
The electronic or molecular mechanisms that initiate photobiomodulation (PBM) in cells are not yet fully understood. The porcine complex III (C-III) of the electron transport chain was characterized with transient absorption spectroscopy (TAS). We then applied our recently developed continuous wave laser coupled TAS procedure (CW-TAS) to investigate the effect of red light irradiances on the heme dynamics of C-III in its c1 reduced state. The time constants were found to be 3.3 ± 0.3 ps for vibrational cooling of the oxidized state and 4.9 ± 0.4 ps for rebinding of the photodissociated axial ligand of the c1 reduced state. The analysis of the CW-TAS procedure yielded no significant changes in the C-III heme dynamics. We rule out the possibility of 635 nm CW light at 4.7 mW/cm2 inducing a PBM effect on the heme dynamic of C-III, specifically with the photodissociation of its axial ligand.
© 2021 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Photobiomodulation (PBM) is the use of low irradiance light in the red to near-infrared (NIR) wavelength range to generate beneficial biological effects in living cells and tissues. Mitochondria, which are the primary organelles involved with ATP production for eukaryotic organisms, are generally accepted as the key targets of PBM therapy [1]. The leading hypothesis for the origin of the PBM effect within the cell is that light modulates the properties associated with the enzyme complexes within the electron transport chain (ETC). Notably, complex IV (C-IV) of the ETC has been a protein of interest among PBM researchers due to the overlap of its absorption spectrum with that of the action spectrum of PBM [2–7].
For the purposes of this study, relatively high concentrations of protein are necessary, and unfortunately C-IV was not available commercially at a concentration higher than 5 mg/mL. In contrast, C-III was commercially available as a lyophilized powder, allowing us to easily make high concentration solutions. Recent work from our group has suggested C-III activity may be altered by low-intensity NIR exposures [8]. While it remains unclear if this is a direct effect on C-III, or is due to indirect effects on C-IV, this makes C-III an appealing target for further study.
C-III has been recently studied with the transient absorption spectroscopy (TAS) technique [9,10]. TAS is a pump-probe spectroscopic technique that can characterize the photophysical dynamics of biological and chemical systems [11]. Though the electronic transitions of C-III have previously been studied via TAS, this work was conducted on proteins from yeast (Saccharomyces cerevisiae) [9] and photosynthetic bacteria (Rhodobacter capsulatus) [10]. TAS characterization of C-III from a mammalian source, such as porcine or bovine, has not been previously conducted. Porcine C-III is highly similar to human C-III (89.54% identity, vs 55.38% and 40.31% identity for the yeast and bacterial C-III respectively) and will be more likely to be predictive of human C-III electronic transitions/time constants, and thus more relevant to human PBM exposures. Therefore, in this study, we focused our efforts on the TAS characterization of C-III from a mammalian source.
Recently, we developed a new technique combining continuous wave (CW) light with TAS, called CW-TAS, for testing samples with light exposures consistent with those found to induce PBM effects [12]. The proof of principle for this technique was established through testing cytochrome c, the heme protein responsible for mediating the electron transfer from C-III to C-IV. No PBM effects were expected when testing with cytochrome c, and indeed the TAS data remained intact and unchanged with the addition of the CW exposures. To further test the CW-TAS technique and to probe potential PBM effects, the more complicated ETC protein, C-III, was tested in this study to investigate if exposures consistent with PBM alter the TAS profile of C-III.
In the present study, we investigate porcine C-III with TAS and characterize the time constants for the oxidized and c1 reduced redox states. This provides the first TAS characterization of C-III specifically from a mammalian source, and we compare the results to previous studies of yeast and bacterial C-III [9,10]. Additionally, we investigate the response of mammalian C-III to irradiances from a CW light source at 635 nm using our previously developed CW-TAS methodology [12]. Since PBM greatly affects mitochondria and its functions [1] and heme proteins are a central part of oxidative phosphorylation, we hypothesize that PBM like changes to C-III will manifest as changes to its transient absorption response, such as production of new transient signals, quenching of existing signals, or changes in the time constants. We prepare C-III in its partially reduced redox state and test it both with and without red CW light exposures during the TAS measurement. By comparing the CW irradiated TAS data to the control, the measured heme dynamics of C-III can be implicated or eliminated as a mechanism underlying the initiation of PBM, leading to a better understanding of the origin of the effect.
2. Methods
2.1. Sample preparation
Complex III from porcine heart was purchased from Sigma-Aldrich (C3381). Complex III (12.5 mg/mL) was prepared in 20 mM KHCO3 (237205, Sigma-Aldrich), pH 8.0 with 20 mM n-dodecyl-β-D-maltoside non-ionic detergent (DDM) (D4641, Sigma-Aldrich). To produce reduced samples of complex III, the sample was prepared in a cuvette sealed with a septum screwcap with a rubber stopper. The sample was purged using gaseous N2 for five minutes. A syringe was then inserted to inject a solution of sodium dithionite (157953, Sigma-Aldrich) to reduce the sample. This reduced form of C-III is referred to in this study as sodium dithionite-treated C-III (SDT-C-III). The oxidation state of all samples was verified by UV-VIS spectroscopy using a Cary 6000i spectrophotometer prior to all TAS experiments. Samples were assayed in a quartz cuvette with a 2-mm path length.
2.2. Transient absorption spectrometer setup
All TAS experiments were carried out using the HELIOS Fire (Ultrafast systems) transient absorption spectrometer described in our previous study [12]. The pump and probe sources were provided by a Ti:sapphire regenerative amplifier (SpitFire Ace, Spectra-Physics) with wavelength centered at 800 nm, pulse duration of 80 fs, and repetition rate of 1 kHz. The pump pulses were generated by splitting part of the 800 nm source light to an optical parametric amplifier (OPA; TOPAS-Twins, Light Conversion), where it was converted to 418 nm light (FWHM ∼8 nm) by the method discussed in previous studies [13,14]. The pump light traveled through a chopper wheel to reduce the repetition rate to 500 Hz. The beam radius of the pump pulse was determined to be about 100 μm at its focal point using the knife-edge technique [15] assuming a 1/e2 beam waist. The probe pulse consisted of a supercontinuum (430-760 nm) generated from the fundamental 800-nm pulse incident upon a sapphire window, also having a repetition rate of 1 kHz. Figure 1 displays a typical supercontinuum generated for the probe pulse.
2.3. Low-irradiance CW laser diode setup
Output from a 635 nm laser diode (CPS635, Thorlabs) was coupled into a fiber imaging system to provide low-power CW irradiation to the sample during TAS scanning. The output power of the diodes was controlled with a continuously variable neutral density filter (NDL-25C-2, Thorlabs) prior to coupling into the 550-μm diameter fiber (M37L02, Thorlabs). The fiber output was imaged to the sample with a flat top beam profile having a diameter of 0.95 cm, resulting in an area of 0.71 cm2 at the cuvette. As detailed previously [12], the fiber output was placed above the pump-probe beam plane, and directed to the cuvette at a shallow angle so that the diode light was not collected at the collection fiber of the HELIOS Fire. The light from the laser diode illuminations did not affect the ΔA(λ, t) spectrum, as verified through observation of the real time ΔA(λ) spectrum in the HELIOS software.
2.4. Testing parameters
For the TAS procedure, the 418 nm pump wavelength was used with a power of 100 μW, and the VIS supercontinuum was used for the probe pulse. The pump and probe pulse overlap was optimized before the start of each TAS measurement by testing with a solution of tetrakis(4-sulfonatophenyl)porphyrin, which is responsive to TAS at our pump wavelength [16], and maximizing the signal response using the Helios spectrometer. For the oxidized C-III, the time delay setup for each scan was programmed to take 0.2-ps steps from -2 to 0 ps, 0.05-ps steps from 0 to 6 ps, 0.2-ps steps from 6 to 25 ps, 1.0-ps steps from 25 to 100 ps, and 25-ps steps from 100 to 7500 ps for a total of 401 time steps. At each time delay step, 0.5 s of measurement were taken, which resulted in 250 ΔA(λ, t) measurements averaged together at that time delay. TAS tests of oxidized C-III were performed without the 635 nm CW exposures, whereas SDT-C-III were performed both with and without the CW exposures.
For the SDT-C-III, the time delay setup for each scan was programmed to take 0.2-ps steps from -2 to 0 ps, 0.05-ps steps from 0 to 3 ps, 0.1-ps steps from 3 to 10 ps, 0.5-ps steps from 10 to 100 ps, and 5-ps steps from 100 to 500 ps for a total of 401 time steps. Similar to the oxidized sample, 0.5 s of measurement were also taken at each time delay step for SDT-C-III. The TAS program was set to take a total of 9 contiguous time scans for each replicate of SDT-C-III tested. For the collections that involved red light irradiances, the first scan was taken as a baseline and did not have any CW laser diode light incident upon the sample. Scans 2-6 were taken with the CW diode emitting light onto the C-III sample with a power density of 4.7 mW/cm2 at the cuvette. Scans 7-9 were taken with the CW diode turned off to observe changes after the full dosage of irradiation [17]. The total time to record 9 scans was 55 min and 47 ± 5 s. The duration of the CW diode exposure during scans 2-6 was computed to be 30 min and 59 ± 3 s. This resulted in an energy density of 8.74 ± 0.02 J/cm2 at the cuvette over scans 2-6. Five replicate data collections were performed with CW-TAS and the red CW laser diode.
2.5. Data processing and analysis
Data was collected and exported from the Helios FIRE software and processed in Surface Xplorer (version 4.3.0, Ultrafast Systems). Background subtractions were performed using the spectra taken at the time delays prior to time-zero. Chirp correction was performed for each data set, and the time-zero correction was acquired by fitting the coherence spiking near the zero delay time in the surface data. CW data sets were processed by averaging together each scan from a replicate sample to the same scan number from other replicate data collections. For data sets without CW exposure, each of the scans within the data collection were averaged together. Global analysis techniques were used to acquire time constants of the excited state dynamics.
Global analysis was conducted in Surface Xplorer by first performing principal component analysis on the surface data through singular value decomposition (SVD) as detailed in previous studies [18]. Time constants were then acquired by fitting the principal components with a sum of exponential decays convolved with a Gaussian instrumentation response function [19], displayed in Eq. (1).
3. Results and discussion
3.1 Absorption spectra of mammalian complex III
Figure 2 presents the ground state absorbance spectra of both oxidized and SDT-C-III. Oxidized C-III had a strong absorption peak in the Soret band at 407 nm. The Soret peak for SDT-C-III was red-shifted with a λmax at 425 nm, and in the Q band, there were peaks at 521 nm and 551 nm. The 418-nm pump’s bandwidth (FWHM ∼8 nm) overlapped with the Soret absorption peaks of both dithionite-treated and oxidized C-III and allows for the excitation of both species.
Fig. 2. Ground state absorbance spectra for C-III measured in its initial oxidized state (black) and after sodium dithionite-treatment (red). Inset plot displays a magnified view of the Q bands.
For the SDT-C-III in this study, the absorbance peaks at 521 nm and 551 nm were indicative of a full reduction of the c1 hemes in the proteins [20]. However, our spectra did not display a second pair of sharp absorbance peaks at ∼530 nm and ∼560 nm, which would be an indicator for the full reduction of the b hemes. When further reduction of the sample was attempted with higher concentrations of sodium dithionite, the proteins formed visible aggregates with no further features in the spectra. However, the 551 nm peak did display a shoulder on its right-hand side which would indicate a partial reduction of the b hemes. This partial, and not full, reduction of the b hemes is quite possibly due to the relatively high concentration of C-III used in this study, and this has similarly been noted in the previous C-III study by Chauvet et al. [9]. We conclude that our dithionite-treated C-III is in a partially reduced state with fully reduced c1 hemes and a partial reduction of the b hemes.
3.2 TAS results of C-III
Transient absorption surface data was collected for samples of oxidized C-III and SDT-C-III and displayed in Figs. 3(a) and 4(a), respectively. For global analysis, the surface data was constrained to the time delay range of -1 to 50 ps and wavelength range of 455-750 nm. The shortened time range was selected to focus on the shorter transients associated with the heme dynamics as opposed to the long-lived transient response. The wavelength range was selected to avoid the pump pulse leakage at shorter wavelengths and the weak probe strength at higher wavelengths. SVD analysis for oxidized C-III yielded singular values of S1 = 0.34, S2 = 0.15, and Sk ≤ 0.07 for k ≥ 3, and the first two spectral (Fig. 3(c)) and kinetic (Fig. 3(d)) components were thus found to be significant and used for global analysis. For SDT-C-III, the singular values were found to be S1 = 0.26 and Sk ≤ 0.04 for k ≥ 2, and the first spectral (Fig. 4(c)) and kinetic (Fig. 4(d)) component was found to be significant and used for global analysis. The principal kinetics were fit using Eq. (1) and for oxidized C-III yielded global time constants of t1 = 0.12 ± 0.03 and t2 = 3.3 ± 0.3 while for SDT-C-III yielded values of t1 = 0.09 ± 0.05, t2 = 0.6 ± 0.1, and t3 = 4.9 ± 0.4. The uncertainty for these values come from the 90% confidence interval of the fit.
Fig. 3. Transient absorption spectra of oxidized C-III with 418 nm excitation at 200 nJ/pulse displayed as (a) a surface and (b) spectral cross sections at the constrained time-delay range used for global analysis. Also provided are the first two (c) spectral components and (d) kinetic components from SVD analysis.
Fig. 4. Transient absorption spectra of SDT-C-III with 418 nm excitation at 200 nJ/pulse displayed as (a) a surface and (b) spectral cross sections at the constrained time-delay range used for global analysis. Also provided are the first two (c) spectral components and (d) kinetic components from SVD analysis.
The global time constants for the ∼3-7 ps heme dynamics (τHeme) of C-III are tabulated in Table 1, along with the previously reported values from earlier studies. The time constants at this timescale, for the reduced states, have been documented to be the recombination time following axial ligand photodissociation [9,10]. For the oxidized state, time constants at this scale have been previously attributed to vibrational cooling [10,21,22]. The τHeme associated with this study’s porcine SDT-C-III is listed to be in the c1 reduced state following the conclusion drawn from its ground state absorption spectra. The SDT-C-III τHeme value obtained from our analysis was close to the τHeme value that was reported for the bacterial C-III and a little faster than the τHeme value reported for the yeast C-III with similar redox states. The oxidized C-III τHeme value was also similar to the value reported in the bacterial C-III study.
Table 1. Global time constant values associated with the heme dynamics (∼5-7 ps) measured in TAS studies on different redox states of C-III.
3.3 CW-TAS results of C-III with 635 nm light
Following the characterization of oxidized C-III and SDT-C-III with the standard TAS procedure, the CW-TAS procedure was performed on n = 5 replicate samples of SDT-C-III. The choice of dosimetry and the 635 nm CW wavelength were meant to approximate exposures expected to generate PBM-like effects, based on results from previous studies [17]. Following data collection and pre-processing, SVD was performed on each of the nine scans of the TAS data taken on SDT-C-III. The wavelength range chosen for global analysis was 455-750 nm. The time delay range was selected to be -1 to 50 ps to focus on the transient responses of 3-7 ps, corresponding to the heme dynamics which were characterized previously with TAS. SVD analysis performed on the CW-TAS data yielded singular values for each scan number of S1 ≥ 0.22 (to a maximum of 0.30), S2 = 0.05, and Sk ≤ 0.03 for k ≥ 3. The first component of the principal kinetics and spectra were thus determined to be the most significant and used for comparative analysis among scan numbers.
Global time constants were computed using the first principal kinetic component of each scan from the CW irradiated collection, and the time constants τHeme corresponding to the ∼5-7 ps heme dynamics are tabulated in Table 2. The τHeme time constant was found to be within the range of 4.7-5.2 ps throughout each of the nine scans. Since the time constants did not deviate outside of the overlap of the confidence intervals, we conclude that the time constant did not change significantly. Because the τHeme time constant remained near the previously obtained SDT-C-III value of 4.9 ± 0.4 ps, and did not approach the oxidized C-III value 3.3 ± 0.3 ps, we conclude that the sample remained in the c1 heme reduced state throughout the CW-TAS procedure. Moreover, the b hemes were not reduced further in any significant way because the time constants did not shift to 6-7 ps based on the values from previous studies [9,10] (see Table 1). Consequentially, this implies that the c1 heme’s ligand photodissociation and recombination process remained to be the dominant process observed throughout the CW-TAS procedure, and the CW light did not induce a significant shift towards vibrational cooling associated with the oxidized state or towards the ligand photodissociation and recombination associated with the b hemes.
Table 2. Global time constants associated with the heme dynamics (∼5-7 ps) for SDT-C-III during the CW-TAS collection with the red CW diode light. Scans were averaged from n = 5 replicate tests, and uncertainty was computed at the 90% confidence interval of the fit.
The first principal spectral component from each of the nine contiguous TAS surfaces are displayed in Fig. 5. For both the control (Fig. 5(a)) and CW-irradiated (Fig. 5(b)), the spectral components did not develop new peaks that would indicate a sharp change in redox state. Relative magnitude changes with progressing scan number were noted among both collections, such as the relaxation of the minima located ∼487 nm and the relaxation at the wavelength range 520-580 nm. Because both the control and CW irradiated data contained these changes, we conclude that the magnitude shifts in the spectra were not the result of a CW induced PBM effect within SDT-C-III and were a naturally occurring change in the samples over time. Comparing with previously obtained first principal spectra from oxidized C-III (Fig. 3(c)) and SDT-C-III (Fig. 4(c)), we correlate the relaxation as a slight shifting of the SDT-C-III spectra towards the oxidized state. Based on these observations, we further conclude that the shifts in magnitude in both the control and CW irradiated data were a result of a slight oxidation of the samples over time. Since the 635 nm CW light is below the energy of the Q-band absorbance peaks for the reduced states of C-III, this selection of CW wavelength was likely too low of energy to stimulate measurable PBM-like effects in our study. Future experiments with C-III should consider a CW wavelength selected from a 520-560 nm range to overlap with an absorbance peak from the reduced c1 or b hemes.
Fig. 5. First principal spectral components from SVD analysis on each of the nine TAS surfaces for the (a) control and (b) CW irradiance collections on SDT-C-III.
4. Conclusions
We have presented the first TAS characterization on C-III specifically from a mammalian source. For porcine C-III, the time constants were found to be 3.3 ± 0.3 ps for vibrational cooling of the oxidized state and 4.9 ± 0.4 ps for rebinding of the photodissociated axial ligand of the c1 reduced state. These time constants are in agreement with previous results obtained on bacterial C-III by Vos et al. [10]. We found that CW-TAS measurements with 635 nm light on SDT-C-III did not induce a change in the time constant τHeme, which implied that the sample remained almost entirely in the c1 reduced state. Based on further analysis of the principal spectra, both with and without CW light, SDT-C-III had undergone a slight oxidation over time in both the control and CW samples. Thus, the slight oxidation was not directly a result of CW light stimulation of the sample. We rule out the possibility of 635 nm CW light at 4.7 mW/cm2 inducing a PBM effect on C-III, specifically with the photodissociation of the axial ligand. The elimination of this electronic process contributes towards a better understanding of PBM by narrowing down the possible electronic or molecular transitions that could be implicated to initiate the PBM effect.
Funding
Army Research Laboratory (W911NF-17-2-0144); National Institutes of Health (1R01GM127696-01, 1R21GM142107); Cancer Prevention and Research Institute of Texas (RP180588); U.S. Department of Defense (W81XWH2010777); Welch Foundation (A-1261); Air Force Office of Scientific Research (19RHCOR067, FA8650-14-D-6519, FA8650-19-C-6024, FA9550-15-1-0517, FA9550-18-1-0141, FA9550-20-1-0366); Office of Naval Research (N00014-20-1-2184); National Science Foundation (CMMI-1826078, DBI-1455671, ECCS-1509268, PHY-2013771).
Acknowledgments
The authors would like to thank SAIC (Airman Systems Directorate, Contracts No. FA8650-14-D-6519 and FA8650-19-C-6024), and the US Air Force Research Laboratory (AFRL). S.P.O. was supported, in part, by the Herman F. Heep and Minnie Belle Heep Texas A&M University Endowed Fund held/administered by the Texas A&M Foundation, the Robert A. Welch Foundation (Grant No. A-1261), and by an appointment to the Student Research Participation Program (Repperger) at AFRL, administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. DoE and AFRL. This research was performed while S.M.P. held an NRC Research Associateship award and N.J.P. was supported by the ORISE Postgraduate Research Participation Program. M.O.S. acknowledges support from the Air Force Office of Scientific Research (AFOSR; FA9550-20-1-0366), the Office of Naval Research (ONR; N00014-20-1-2184), the Robert A. Welch Foundation (Grant No. A-1261), and the National Science Foundation (NSF; PHY-2013771). M.L.D. acknowledges support from AFOSR (19RHCOR067). V.V.Y. acknowledges partial funding from NSF (DBI-1455671, ECCS-1509268, CMMI-1826078), AFOSR (FA9550-15-1-0517, FA9550-18-1-0141, FA9550-20-1-0366), Army Research Laboratory (W911NF-17-2-0144), DOD Army Medical Research (W81XWH2010777), ONR (N00014-16-1-2578), NIH (1R01GM127696-01,1R21GM142107), and the Cancer Prevention and Research Institute of Texas (RP180588).
Distribution Statement A
Approved for public release; distribution is unlimited. PA# 502ABW-2021-0041. The opinions expressed in this document, electronic or otherwise, are solely those of the author(s). They do not represent an endorsement by or the views of the United States Air Force, the Department of Defense, or the United States Government.
Disclosures
The authors declare no conflicts of interest.
Data availability
Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the authors upon reasonable request.
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