Kinetic Determination of Cytochrome b₆f Activity In Vitro

Materials and reagents

Biological materials

1. The purified complexes used in this study (PSI, Cytb₆f, PC, and FDX) were isolated from green algae following the specific protocols described in [17]. These procedures were followed exactly as described without any further modifications. Each protein fraction was verified for purity and activity via absorbance spectroscopy and SDS-PAGE prior to being aliquoted and stored at -80 °C.

Reagents

1. 3-(Morpholin-4-yl)propane-1-sulfonic acid (MOPS) (Sigma, catalog number: 106129)

2. Antimycin A (AA) (Sigma, catalog number: A8674)

3. Ascorbate (Asc) (Sigma, catalog number: 255564)

4. Hydrochloridic acid (HCl) (Roth, catalog number: 7647-01-0)

5. Magnesium chloride (MgCl₂) (Sigma, catalog number: M8266)

6. Potassium chloride (KCl) (Sigma, catalog number: P9541)

7. Trans-PCC α-maltoside (t-PCCαM) (Glycon, catalog number: D99019)

8. Tricine (C₆H₁₃NO₅) (AppliChem, catalog number: A10585)

9. Water (ddH₂O), in-house Millipore-purified water (18.2 MΩ/cm)

Solutions

1. MOPS stock, pH 7.0, 1 M (see Recipes)

2. Tricine stock, pH 7.8, 1 M (see Recipes)

3. KCl stock, 3 M (see Recipes)

4. Ascorbate stock, 1 M (see Recipes)

5. MgCl₂ stock, 1 M (see Recipes)

6. t-PCCαM stock, 10% (see Recipes)

7. JTS activity solution (JAs) (see Recipes)

8. Cytb₆f purification solution (CPs) (see Recipes)

9. PSI purification solution (PPs) (see Recipes)

10. Plastocyanin column buffer (PC-B) (see Recipes)

Recipes

1. MOPS stock, pH 7.0, 1 M

a. Set pH to 7.0 using HCl 5 M.

b. Keep cool (4 °C) to avoid contaminations; life span ~6 months.

2. Tricine stock, pH 7.8, 1 M

a. Set pH to 7.8 using HCl 5 M.

b. Keep cool (4 °C) to avoid contaminations; life span ~6 months.

3. KCl stock, 3 M

Keep at room temperature (24 °C); life span unlimited.

4. Ascorbate stock, 1 M

Keep in a dark tube. It is better to use a fresh solution mixed at the day of experimenting; short lifespan.

5. MgCl₂ stock, 1 M

Keep cool (4 °C) to avoid contamination; life span unlimited.

6. t-PCCαM stock, 10%

Dissolves better in hot water. Aliquot to 1 mL tubes and keep at -20 °C to increase lifespan (~6 months).

7. JTS activity solution (JAs)

Keep cool (4 °C) to avoid contaminations; life span 2–3 months.

8. Cytb₆f purification solution (CPs)

Keep cool (4 °C) to avoid contaminations; life span 2–3 months.

9. PSI purification solution (PPs)

Keep cool (4°C) to avoid contaminations; life span 2–3 months.

10. Plastocyanin column buffer (PC-B)

Keep cool (4 °C) to avoid contaminations; life span ~6 months.

Equipment

1. Joliot type spectrophotometer -150 (Bio-Logic) designed for fast kinetics and simultaneous multi-wavelength characterization.

a. Smart lamp (detection): multi-LED probing source with integrated interference filters; 546, 554, 563, 574, 705, and 740 nm wavelengths used (standard kit for cytochrome b₆f and P700); pulsed LED measurements can be acquired as fast as 10 μs between samples.

b. Actinic light source, type: dual-ring LED array (integrated into the optical bench); output: 630 nm (red) and 720 nm (far-red); intensity range: 0 to >5,000 μE/m²/s¹.

c. Detection system: Detectors: two large-area high-speed Si PIN photodiodes (signal and reference); resolution: 18-bit ADC for high dynamic range; sensitivity: capable of resolving absorbance changes down to 10 × 10^-5 OD.

d. Synchronization: Integrated BNC ports for microsecond-precision triggering of the Nd:YAG laser and external flash lamps.

2. Laser flash (Quantel, Lumibird Group), Nd:YAG (neodymium-doped yttrium aluminum garnet) solid-state laser; primary output: frequency-doubled to 532 nm (green); wavelength shifting: the 532 nm beam is used to pump a red dye filter/cavity (e.g., using LDS 698 dye); effective output wavelength: saturating red flash cantered at ~640–700 nm; pulse duration: ultra-short pulses of ~6–10 ns; pulse energy (strength): calibrated to ~20–30 mJ at the source.

Experimental rationale: The energy is adjusted to ensure a single saturating turnover of all photosystem I (PSI) reaction centers within the cuvette while the red shift prevents spectral interference with the JTS detection wavelengths.

Procedure

A. Sample preparation and composition

1. Perform assays in a 1 mL quartz cuvette with a final working volume of 400 μL in JAs buffer, supplemented with ascorbate 10 mM, MgCl₂ 2.5 mM, and KCl 7.5 mM. The basal reaction mixture contains isolated PSI (250 nM, calibrated per reaction center), isolated Cytb₆f (2.13 μM), and PC (recombinant) (1 μM).

Notes:

1. For each biological replicate, a control measurement was performed in the absence of Cytb₆f. This served to isolate the P700 signature and confirm that the green region absorbance changes were specifically attributable to the cytochrome complex.

2. The concentration of photosystem I (PSI) was determined via chlorophyll quantification in 80% acetone. To ensure accurate stoichiometry within the reconstituted system, the PSI concentration was adjusted to your target concentration, e.g., 50 nM, of reaction centers (RC), based on the established ratio of 217 Chl per PSI RC for Chlamydomonas reinhardtii. This calibration allows for the precise determination of electron transfer rates per functional unit.

2. To elucidate the inter-complex electron transfer kinetics, add plastocyanin (PC) and ferredoxin (FDX) sequentially at the following concentrations: FDX titration, 0.5, 1, 2, and 4 μM; PC titration: 2, 3, 4, and 5 μM.

B. Spectroscopic setup (JTS-150 configuration)

1. To simultaneously conduct all detections, use the Biologic "Smart Lamp" LED system, allowing for rapid automated switching between discrete wavelengths.

2. Monitor absorbance changes at 546, 554, 563, 573, 705, and 740 nm.

Notes:

1. 554 nm and 563 nm target Cytf and Cytb, respectively, while 705 nm monitors P700 redox states.

2. 546/573 nm serve as isosbestic/reference points for Cytf and Cytb, while 740 nm serves as a reference for P700.

3. To enable simultaneous measuring of both the green (540–580 nm) and far-red (700⁺ nm) regions, remove all physical filters from the detector path.

Note: Although orange actinic light (350 μE/m²/s¹) was present, the JTS-150 detectors utilize pulsed-probe technology (see Biologic product page for further details). Since the detector only integrates the signal during a specified measurement window, which is synchronized with the flashes of the measuring “Smart Lamp,” the continuous background actinic light is treated as a constant offset and subtracted, preventing sensor saturation or interference with kinetic data.

4. At the beginning of the initial measurement, you are required to balance the detector signal. Here, set the balance to “Automatic” and set all detection values to 5 V.

Note: To improve signal/noise ratio, twist the smart lamp to facilitate an increased signal voltage for the reference measurements of 554 nm and (if applicable) 705 nm.

Critical: When balancing, make sure that no reference detection surpasses the maximal voltage (10 V).

C. Measurement procedure and sequence architecture

Note: Each measurement consisted of four technical replicates per test to ensure statistical robustness and decrease signal/noise ratio. The sequence is structured into two distinct phases: laser-induced flash kinetics and steady-state actinic flux, followed by dark relaxation (Figure 2).

1. To resolve the rapid electron transfer between Cytb₆f and P700, apply a series of four laser flashes.

a. Use the first flash to verify laser stabilization and adequate signal.

b. Implement an initial delay of 700 μs to exceed the measurement resolution. A critical delay of 2 ms must be maintained between the laser flash and the first probe pulse.

Note: The 2 ms delay is strictly dictated by the mechanical constraints of the laser shutter and light diffraction within the cuvette. In the absence of specialized P700 interference filters (removed to allow multi-wavelength green/red detection), shorter delays resulted in diffracted laser light saturating the detector. The initial detection can vary between systems.

2. Following the laser phase, subject the sample to a dark pause to reset the redox states before driving the full inter-complex chain.

a. Dark pause: 2 s.

b. Actinic phase: 2 s of continuous actinic light to reach steady-state turnover.

c. Recovery phase: 20 s of detection in darkness to monitor the re-reduction of P700 and the oxidation/reduction cycles of the Cytb₆f pool.

3. Use the following sequence command:

20(50msD)T4(10(200msD)A[on]10usA[off]140usB[on]10usB[off]2000usD10(200msD)3(20(50msD)A[on]10usA[off]140usB[on]10usB[off]1140us{700us,100,5s,D})10(200msD)20(50msD){1200us,100,2s, H350usD50usG[8000uE]}H{1200us,200,20s,D})

This sequence can be summarized into the segments shown in Table 1.

Figure 2. Exemplary trace. The measurement includes four technical replicates. Each begins with a probe-laser flash to initialize laser activation, followed by three laser flashes. Flowing a relaxation period, actinic light (orange ring, 350 μE/m²/s¹) is activated for 2 s, followed by 20 s of darkness. To simplify, we present here only the measured absorbance at 705 nm for a sample containing the experimental initial setup (including Cytb₆f and PSI), accompanied by FDX (4 μM) and PC (4 μM).

Table 1. Sequence breakdown segmentation

D. Data analysis and signal processing

Data acquisition is performed using the JTS-150 proprietary software (Bio-Logic). However, while the provided software is capable of performing all necessary deconvolutions and kinetic calculations (and users are advised to refer to the manufacturer’s manual for these standard procedures), the calculations in this protocol were performed manually in Microsoft Excel using the formulas listed below. This approach was taken to expand the analytical view and to validate that the resulting traces were consistent with the original protocol and established spectral coefficients. Data analysis and curve fitting (kinetic deconvolution and rate constant determinations) were performed using Origin.Pro 2024 (OriginLab Corporation, Northampton, MA, USA). To mimic the same analysis, follow the following steps:

1. Spectral deconvolution and baseline correction: To isolate the signals for Cytf and Cytb, perform a linear baseline correction using two reference wavelengths (546 nm and 573 nm) where cytochrome absorbance is not dependent on its reduction state.

a. Initial Cytf correction: Since there is some overlap between the absorbance of the heme groups, to eliminate the effects of Cytb on the calculated Cytf trance, first correct the trace by subtracting 27% of the measured detection for 563 nm, as discussed in [18], by using the following formula:

${D^{*}}_{554}=D_{546}-0.27\times (D_{563}-D_{546})$

b. Signal correction: For every detection point, calculate a linear slope between the optical densities (D) at 546 and 573 nm. The corrected baseline value (D_corr) for a target wavelength is determined as follows:

$W{L}_{corr}=WL\times \left(\frac{D_{546}-D_{573}}{546-573}\right)+(D_{546}-546\times \left(\frac{D_{546}-D_{573}}{546-573}\right))$

Where D are the detections at a given wavelength, and WL could be either 554 for Cytf or 563 for Cytb.

c. Signal isolation: The specific redox signal can then be calculated as:

Cytf = ${D^{*}}_{554}$ – 554_corr

Cytb = D₅₆₃ – 563_corr

P700 Kinetics: The P700 signal is isolated by a simple differential subtraction, as follows:

P700 = D₇₀₅ – D₇₄₀

The resulting traces and the correction methodology are illustrated in Figure 3.

Figure 3. Assembled kinetic data. (a) Absorbance changes of isolated photosystem I complexes, following a laser flash, were detected for 5 s (here, the first second is presented). The result shows the averaged data of 12 technical replicates per trace. Tests examined the re-reduction rate with increasing FDX concentrations (red gradient), which showed no change. Following that, an increasing concentration of PC was added to the tested mixture (blue gradient). The effect of actinic light (orange ring, 350 μE/m²/s¹) was examined then. (b) Four green wavelengths were detected at 546 nm (yellow), 573 nm (orange), 563 nm (red), and 554 nm (black). Signal correction was applied for Cytf reduction kinetic determination (dashed blue), and the trace for Cytf oxidation/reduction was calculated (dotted blue). Such calculations were conducted with all previously specified tests. (c) Increasing FDX concentrations (red gradient) showed no changes in the first-order kinetics (see arrow) and diminished steady-state oxidation of Cytf (following 2 s of illumination). (d) On the other hand, increasing concentrations of PC increase the first-order kinetics rate, which results in an increased steady-state oxidation.

2. Generate an average of 12 measurements (3 data flashes per 4 technical replicates) to enhance the signal/noise ratio Cytf, Cytb, and P700 (Figure 3). Following the laser flash, P700⁺ re-reduction typically displays two phases:

a. First-order phase: Represents electron transfer from PC already pre-bound to PSI in a complex before the flash. With a half-life of approximately 4 μs [19], this phase is below our detection resolution, due to the imposed delay (2 ms).

Note: We can assess it by looking at the initial amplitude of the trace; since the first detection is past the initiation of the second phase, it will be composed of a mixture of the two.

b. Second-order phase: Represents the binding of reduced, free-diffusing PC to PSI. The second-order rate is sensitive to the kinetic properties and concentrations of both PSI and PC [17].

Note: A high affinity between PC and Cytb₆f may decrease the apparent concentration of free PC available for PSI reduction, slowing the observed rate.

3. Data from 4 technical replicates are averaged. The analysis focuses on the window starting 1 s before AL, the 2-s AL period, and 2 s of subsequent dark relaxation (Figure 3).

AL Phase (Steady State): Cytf and P700 kinetics are fitted using a double exponential decay.

a. Fast phase: t_1/2 ~10 ms, reflects the initial rapid turnover.

b. Slow phase: t_1/2 ~500 ms, reflects the transition toward a steady-state equilibrium.

c. Dark phase (post-AL): Upon turning off the light, Cytf re-reduction follows a single exponential decay. This rate is attributed to ferredoxin (FDX)-mediated re-reduction.

E. Additional perspectives and future applications

Mechanism for ferredoxin- cytochrome b₆f reduction: A key uncertainty remains on whether FDX reduces Cytf directly or via the Cytb hemes. In general, the redox potential of FDX is high enough to reduce all heme groups, and the specific site where it interacts with Cytb₆f in this isolated system is not clear (as discussed in [17]). Further research is required on the subject, and we urge the scientific community to pursue this topic. Our proposed electron flow scheme, including the potential bypasses or direct reduction pathways, is shown in the Graphical overview.

The reduction state of Cytb is generally studied via spectroscopic evaluation of the absorbance changes at 563 nm. However, here we encounter two issues:

a. The absorbance changes of Cytb are not as prominent as Cytf since, in this experimental setup, the primary electron donor for Cytb (i.e., plastoquinol) is absent.

b. There is a grave overlap between Cytf and Cytb absorbance change, which could technically overshadow differences observed at 563 nm.

For these reasons, at this point, we cannot undisputedly claim that the observed absorbance changes can reflect the reduction state of Cytb. Further insight into the matter would require additional investigations, which we hope to cover in future studies.

This protocol provides a template for exploring further interactions within the photosynthetic chain. Future studies using this setup may include:

a. FNR interactions: Testing the impact of Ferredoxin-NADP⁺ Oxidoreductase on the re-reduction kinetics., as was proposed by [20,21].

b. Regulatory proteins: Assessing how auxiliary proteins such as STT7 [22], pgr5 [23], or PetP analogs [24] influence the Cytb₆f complex under different physiological states.

c. Environmental interface studies: Testing the effects of environmental conditions (pH, salinity, etc.) on complex formation between Cytb₆f and PC.

d. Inhibitor studies: Characterizing blockades on Cytb₆f activity using specific inhibitors such as:

i. Antimycin A: postulated to target the Qi site on the stromal side [18].

ii. DBMIB: blocking the Rieske FeS to Cytf transfer) [25].

iii. DNP-INT: inhibits PQH₂ oxidation at the Qo site [25].

Validation of protocol

This protocol or parts of it has been used and validated in the following research articles:
•  Milrad et al. [17]. Insights into plastocyanin–cytochrome b6f complex formation: The role of plastocyanin phosphorylation. Plant Physiol.

General notes and troubleshooting

We list here the potential issues that may arise from using slightly different instrumentation or minor deviations from the protocol.

Acknowledgments

Conceptualization, Y.M, M.H.; Investigation, Y.M., D.W.; Writing—Original Draft, Y.M.; Writing—Review & Editing, Y.M., M.H.; Funding acquisition, Y.M., M.H.; Supervision, M.H.

This research was financed by the Deutsche Forschungsgemeinschaft (DFG HI739/13-1-3 DFG/9-1/-2, and 25-1 in DFG FOR 5573/1), in association with the RECTOR Program of the University of Okayama, Japan. Y.M. acknowledges the Alexander von Humboldt Foundation for a Research Fellowship for postdocs (1219125).

The authors would like to acknowledge the contribution of Felix Buchert and Michael Vaughn for their technical support in setting up the instrumentation and for providing insights on the spectroscopic aspects of this research.

This protocol was described and validated in [17]. The presented figures were created using BioRender by Milrad, Y and are publicly available using the following link: https://BioRender.com/hf2cag3.

Competing interests

The authors declare no conflicts of interest.

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