Recombinant Synechococcus elongatus Cytochrome b559 subunit alpha (psbE)

What is Cytochrome b559 and what role does the alpha subunit (psbE) play in Synechococcus elongatus?

Cytochrome b559 is an essential component of photosystem II (PSII) in photosynthetic organisms, including the cyanobacterium Synechococcus elongatus. It consists of alpha (psbE) and beta (psbF) subunits that together form a heterodimer with a non-covalently bound heme group. The alpha subunit provides critical histidine residues for heme ligation and participates in maintaining PSII stability and function. Studies have demonstrated that Cytochrome b559 plays dual roles in both the assembly/stability of PSII and photoprotection mechanisms against photoinhibition . In mutant studies, alterations to the alpha subunit resulted in increased susceptibility to photoinhibition, highlighting its importance in photoprotective functions .

Why is Synechococcus elongatus PCC 7942 a preferred model organism for studying recombinant photosynthetic proteins?

Synechococcus elongatus PCC 7942 has become an excellent synthetic biology chassis and model system for several compelling reasons. First, it possesses a relatively small genome (approximately 2.7 Mb) with 55.5% GC content, making it genetically tractable . Second, it can be easily manipulated through natural transformation or conjugation from E. coli, facilitating efficient genetic modifications . Third, it serves as an important model for studying prokaryotic circadian rhythms, nutrient regulation, environmental responses, and lipid metabolism . Additionally, of the organism's 2,723 genes, only 718 have been identified as essential for survival under laboratory conditions, providing flexibility for genetic manipulation without compromising viability .

What are the different redox forms of Cytochrome b559 observed in Synechococcus elongatus, and what is their significance?

Cytochrome b559 exists in multiple redox forms, including high-potential (HP) and low-potential (LP) forms. Research with mutant strains has revealed that modifications to the alpha subunit can significantly alter the distribution of these redox forms. For instance, H22Kα and Y18Sα mutants in Synechocystis sp. PCC 6803 (comparable to Synechococcus systems) predominantly contained the oxidized LP form of Cytochrome b559 (approximately 79% and 86%, respectively) . The redox potential of Cytochrome b559 is strongly influenced by the hydrophobicity and ligation environment of the heme, with specific arginine residues in close contact with heme propionates affecting the electrostatic interactions and consequently the redox properties . These different redox forms are significant as they correlate with the photoprotective function of Cytochrome b559, with mutants exhibiting altered redox states showing higher susceptibility to photoinhibition under strong light conditions .

How do site-specific mutations in the psbE gene affect photosystem II assembly and photoprotection mechanisms in Synechococcus elongatus?

Site-specific mutations in the psbE gene have revealed critical insights into photosystem II (PSII) function. Studies on comparable cyanobacterial systems have shown that mutations affecting key residues of the alpha subunit can significantly impact both PSII assembly and photoprotection. For example, H22Kα and Y18Sα mutations (corresponding to Y19Sα in T. elongatus) resulted in predominately low-potential (LP) forms of Cytochrome b559 in PSII core complexes . These mutations demonstrated functional yet compromised PSII, exhibiting normal period-four oscillation in oxygen yield but significantly increased susceptibility to photoinhibition under high-light conditions .

A particularly revealing finding was that when the same mutations were introduced in a D1-D170A genetic background (which prevents assembly of the manganese cluster), they almost completely abolished PSII accumulation even under normal light conditions . This suggests a critical redox role of Cytochrome b559 that becomes essential when the oxygen-evolving complex is compromised. The precise molecular mechanisms involve altered ligation structures and redox properties of the heme in Cytochrome b559, as electrostatic interactions between arginine residues (specifically Arg8 and Arg18 of the α-subunit) and the heme propionates are disrupted by these mutations .

What transcriptomic and metabolomic changes occur following integration of recombinant constructs into the Synechococcus elongatus genome?

Integration of recombinant DNA constructs into the Synechococcus elongatus genome can trigger substantial transcriptomic and metabolomic changes. As demonstrated in comparable studies, genomic integration can significantly alter energy metabolism and carbon fixation pathways . Transcriptomic analyses have revealed that integration events can activate or repress numerous genes, with particular impacts on photosynthesis-related pathways .

For example, when foreign genomic elements were integrated into S. elongatus PCC 7942 via homologous recombination, researchers observed decreased photosynthesis and carbon fixation compared to control strains . Metabolomic analyses further confirmed these alterations, revealing shifts in carbon flux, energy production pathways, and cellular homeostasis mechanisms . These changes highlight the importance of comprehensive multi-omics analyses when working with recombinant Synechococcus strains, as modifications intended to study one protein (e.g., psbE) may have cascading effects throughout metabolic networks.

How does the interaction between psbE (Cytochrome b559 alpha subunit) and other photosystem II components change under various environmental stressors?

The interaction between psbE and other photosystem II components demonstrates remarkable plasticity under various environmental stressors. Research has shown that under high-light conditions, the photoprotective function of Cytochrome b559 becomes crucial, with its redox state shifting in response to excess excitation energy . Mutants with altered psbE, such as H22Kα and Y18Sα variants, exhibit significantly slower recovery of oxygen-evolving activity after photoinhibition compared to wild-type strains .

A notable aspect of these interactions is their dependence on specific photosystem II isoforms. For instance, in T. elongatus, the PsbA3 isoform of D1 protein is specifically expressed under high-light conditions and interacts differently with Cytochrome b559 compared to other D1 isoforms . This suggests a coordinated stress response mechanism where psbE function is modulated by compositional changes in interacting proteins. Furthermore, the manganese cluster assembly appears intricately linked to Cytochrome b559 function, as demonstrated by the severe PSII assembly defects in double mutants affecting both systems .

What are the comparative differences in Cytochrome b559 structure and function between Synechococcus elongatus and other model cyanobacteria like Synechocystis sp. PCC 6803?

Comparative analysis of Cytochrome b559 between Synechococcus elongatus and other model cyanobacteria reveals both conserved features and species-specific adaptations. While the core structure and function remain similar across cyanobacterial species, subtle differences exist in amino acid sequences, redox properties, and regulatory mechanisms.

What are the most effective methods for transformation and expression of recombinant psbE in Synechococcus elongatus PCC 7942?

The most effective methods for transformation and expression of recombinant psbE in Synechococcus elongatus PCC 7942 leverage the organism's natural competence and homologous recombination capabilities. For optimal transformation, researchers should:

• Select the appropriate transformation method: For large DNA constructs (>30kb), triparental conjugative transfer is superior to natural transformation . This involves using helper plasmids such as pRL443 and pRL623 to facilitate DNA transfer from E. coli to Synechococcus .

• Use log-phase cultures: Transformation efficiency is highest when Synechococcus cultures are in logarithmic growth phase (OD750 of 1-2) .

• Optimize DNA quality and concentration: Use high-quality, supercoiled plasmid DNA prepared with commercial purification kits for maximum transformation efficiency .

• Target neutral integration sites: The neutral site 1 (NS1) locus has been developed specifically for integration of foreign genes without disrupting essential functions .

• Employ codon optimization: Given the high GC content (~55.5%) of the Synechococcus genome, expression levels are significantly improved when the gene of interest is adapted to the preferred codon usage patterns (1st letter GC ~64%, 2nd letter GC ~44%, 3rd letter GC ~60%) .

• Use appropriate vectors: Vectors like pSyn_6 (4461 bp) are designed specifically for Synechococcus expression and include features such as NS1 homologous recombination sites, the strong constitutive psbA promoter, and options for N-terminal and C-terminal tags .

• Perform transformation in darkness: Incubating transformation mixtures at 34°C in the dark increases transformation efficiency .

• Maintain proper selective pressure: Use appropriate antibiotics (e.g., spectinomycin at 50-100 μg/mL) for selection of transformants .

How should researchers design experiments to investigate the effects of psbE mutations on photosystem II efficiency and photoprotection?

Designing robust experiments to investigate psbE mutations requires a multi-faceted approach that combines genetic engineering, physiological measurements, and biochemical analyses:

• Mutation design strategy:

  • Target conserved residues known to affect heme ligation (e.g., His22) or redox potential (e.g., Arg8, Arg18)

  • Create a variety of mutation types: conservative substitutions, charge inversions, and deletions

  • Design mutations that specifically affect different properties: heme binding, protein-protein interactions, or redox potential

• Genetic manipulation approach:

  • Use site-directed mutagenesis to create precise mutations in the psbE gene

  • Employ homologous recombination-based transformation techniques

  • Construct strains with mutations in both psbE and other photosystem components (e.g., D1 protein) to study combinatorial effects

• Physiological measurements:

  • Oxygen evolution measurements under varying light intensities

  • Chlorophyll fluorescence analysis (OJIP transients, Fv/Fm ratios)

  • Growth rate comparisons under different light regimes

  • Recovery kinetics following high-light exposure

• Biochemical analyses:

  • Spectroscopic characterization of Cytochrome b559 redox states

  • Determination of the ratio between high-potential and low-potential forms

  • Analysis of protein complex stability using native gel electrophoresis

  • Photosystem II isolation and activity assays

• Experimental controls:

  • Include wild-type strains processed identically to mutants

  • Generate complementation strains to confirm phenotype causality

  • Use multiple independent mutant lines to rule out secondary mutations

• Environmental variables:

  • Test under standard growth conditions (34°C, continuous illumination at 50-100 μmol photons m⁻² s⁻¹)

  • Expose to high-light stress (500-1000 μmol photons m⁻² s⁻¹)

  • Evaluate performance under fluctuating light conditions

  • Test recovery capabilities after photoinhibition

These experimental designs should incorporate proper statistical analysis with multiple biological and technical replicates to ensure reproducibility and significance of the observed effects.

What protocols are recommended for isolating and characterizing recombinant Cytochrome b559 from Synechococcus elongatus?

Isolation and characterization of recombinant Cytochrome b559 from Synechococcus elongatus requires specialized protocols that preserve protein integrity while achieving high purity. The following comprehensive approach is recommended:

Isolation Protocol:

• Cell Culture and Harvesting:

  • Grow Synechococcus elongatus transformants in BG-11 medium at 34°C under continuous illumination (50-100 μmol photons m⁻² s⁻¹) with 1-2% CO₂ in air

  • Harvest cells in late exponential phase (OD₇₅₀ = 2.0-3.0) by centrifugation at 6,000 × g for 10 minutes at 4°C

  • Wash cell pellet twice with buffer containing 50 mM HEPES-NaOH (pH 7.5), 10 mM MgCl₂, and 5 mM CaCl₂

• Cell Disruption:

  • Resuspend cells in lysis buffer (50 mM HEPES-NaOH pH 7.5, 10 mM MgCl₂, 10% glycerol, 1 mM PMSF, 1 mM benzamidine, and 1 mM ε-aminocaproic acid)

  • Disrupt cells using either French pressure cell (20,000 psi) or bead-beating with 0.1 mm glass beads

  • Remove unbroken cells by centrifugation at 5,000 × g for a single target cyanobacterial protein (such as psbE gene products)

  • Add 1 mM AEBSF and 1× protease inhibitor cocktail if implementing partial purification of membrane protein complexes

• Membrane Isolation:

  • Separate thylakoid membranes by ultracentrifugation at 100,000 × g for 60 minutes at 4°C

  • Resuspend membrane pellet in buffer containing 50 mM MES-NaOH (pH 6.5), 10 mM MgCl₂, 5 mM CaCl₂, and 25% glycerol

  • For histidine-tagged constructs, add imidazole to 5 mM final concentration

• Solubilization and Purification:

  • Solubilize membranes with 1% n-dodecyl-β-D-maltoside (β-DDM) or 1% digitonin at a detergent:chlorophyll ratio of 20:1 for 30 minutes on ice

  • Clear the solution by centrifugation at 20,000 × g for 20 minutes

  • If using histidine-tagged constructs (from the pSyn_6 vector system), purify using Ni-NTA affinity chromatography with increasing imidazole concentrations (20-250 mM)

  • For native protein, use ion exchange chromatography followed by size exclusion chromatography

Characterization Methods:

• Spectroscopic Analysis:

  • UV-visible spectroscopy to determine the absorption spectra of oxidized and reduced forms (characteristic peaks at 559 nm in reduced state)

  • Difference spectroscopy to quantify the high-potential and low-potential forms

  • Electron paramagnetic resonance (EPR) to analyze the redox properties and coordination environment of the heme

• Functional Assays:

  • Redox potential determination using potentiometric titrations

  • Oxygen evolution measurements of reconstituted systems

  • Electron transfer kinetics analysis using flash photolysis

• Structural Characterization:

  • Circular dichroism to assess secondary structure

  • Mass spectrometry for accurate molecular weight determination and post-translational modifications

  • If possible, X-ray crystallography or cryo-EM for high-resolution structural information

• Protein-Protein Interaction Analysis:

  • Co-immunoprecipitation with other photosystem II components

  • Crosslinking studies to identify interaction partners

  • Native gel electrophoresis to assess complex formation

This systematic approach ensures reliable isolation and comprehensive characterization of recombinant Cytochrome b559 from Synechococcus elongatus.

How can researchers effectively integrate recombinant psbE into neutral sites of the Synechococcus elongatus genome?

Effective integration of recombinant psbE into neutral sites of the Synechococcus elongatus genome requires careful vector design and transformation optimization. The following detailed methodology ensures successful genomic integration:

• Vector Design Considerations:

  • Utilize vectors containing neutral site 1 (NS1) homologous recombination sequences flanking the gene of interest

  • Include a strong constitutive promoter such as the psbA promoter for high-level expression

  • Incorporate an appropriate antibiotic resistance marker (e.g., spectinomycin resistance) for selection

  • Consider adding N-terminal or C-terminal tags (His-tag, V5 epitope) for detection and purification if needed

  • Ensure the gene of interest is codon-optimized for Synechococcus elongatus' high GC content (~55.5%)

• Preparation of High-Quality Vector DNA:

  • Produce supercoiled plasmid DNA using commercial kits like PureLink™ HQ Mini Plasmid Purification kit

  • Maintain DNA concentration between 50-100 ng/μL for optimal transformation

  • Verify plasmid integrity by restriction digest and sequencing

• Transformation Protocol:

  • Culture Synechococcus elongatus PCC 7942 in BG-11 medium at 34°C until reaching log phase (OD₇₅₀ of 1-2)

  • Harvest 1.5 mL of cells by centrifugation at 14,000 rpm for 3 minutes

  • Wash cells with fresh BG-11 medium

  • Resuspend in 100 μL BG-11 medium

  • Add 100 ng of supercoiled plasmid DNA and incubate at 34°C in the dark for 24 hours

  • Plate on BG-11 agar containing spectinomycin (50 μg/mL)

  • Incubate plates at 34°C under continuous illumination (50 μmol photons m⁻² s⁻¹) for 7-14 days until colonies appear

• Verification of Integration:

  • Screen transformants by colony PCR using primers that span the integration junction

  • Confirm full integration and plasmid backbone loss by PCR

  • Verify expression of the recombinant protein by Western blot

  • Assess phenotypic effects through growth and photosynthetic activity measurements

• Segregation Analysis:

  • Restreak colonies on selective media 3-4 times to ensure complete segregation

  • Confirm homozygosity of the genome modification by PCR

  • Quantify copy number if necessary using quantitative PCR

This approach leverages the natural transformability of Synechococcus elongatus and its homologous recombination machinery to achieve precise genomic integration at neutral sites, minimizing disruption to essential cellular functions while enabling controlled expression of the recombinant psbE gene.

What are common pitfalls in expressing recombinant Cytochrome b559 in Synechococcus elongatus, and how can researchers address them?

When expressing recombinant Cytochrome b559 in Synechococcus elongatus, researchers frequently encounter several challenges that can compromise experimental outcomes. Here are the most common pitfalls and their solutions:

By anticipating these common pitfalls and implementing the suggested solutions, researchers can significantly improve the success rate of recombinant Cytochrome b559 expression studies in Synechococcus elongatus.

How can researchers troubleshoot unexpected phenotypes in Synechococcus elongatus strains expressing mutant forms of psbE?

Unexpected phenotypes in Synechococcus elongatus strains expressing mutant forms of psbE require systematic troubleshooting to determine the underlying mechanisms. The following comprehensive approach helps researchers identify and resolve issues:

• Verify Genetic Construction:

  • Issue: Unintended mutations or integration errors.

  • Approach: Perform whole-genome sequencing or targeted sequencing of the mutant construct and flanking regions. Verify that no additional mutations were introduced during transformation. Check for complete segregation of the mutation using PCR.

• Analyze Protein Expression and Stability:

  • Issue: Altered protein levels or stability.

  • Approach: Quantify protein accumulation using Western blotting with specific antibodies. Compare transcript levels using RT-qPCR. Assess protein half-life through pulse-chase experiments. Examine protein localization using fractionation or fluorescent tagging.

• Characterize Photosynthetic Parameters:

  • Issue: Unexpected changes in photosynthetic function.

  • Approach: Measure oxygen evolution under different light intensities. Perform chlorophyll fluorescence measurements (Fv/Fm, NPQ, electron transport rate). Analyze P700 oxidation-reduction kinetics. Compare growth rates under various light regimes.

• Investigate Redox Properties:

  • Issue: Altered redox behavior of Cytochrome b559.

  • Approach: Determine the ratio of high-potential and low-potential forms using spectroscopic methods . Perform potentiometric titrations to precisely measure redox potentials. Compare susceptibility to redox-active compounds.

• Assess Protein-Protein Interactions:

  • Issue: Disrupted interactions with other photosystem components.

  • Approach: Perform co-immunoprecipitation experiments. Use Blue Native-PAGE to examine complex formation. Conduct crosslinking studies followed by mass spectrometry to identify altered interaction partners.

• Examine Stress Responses:

  • Issue: Unexpected sensitivity to environmental stressors.

  • Approach: Challenge mutant strains with high light, oxidative stress, temperature fluctuations, and nutrient limitations. Compare recovery kinetics after photoinhibition . Analyze expression of stress-responsive genes.

• Conduct Comprehensive -Omics Analysis:

  • Issue: System-wide effects beyond the immediate mutation.

  • Approach: Perform transcriptomics to identify compensatory gene expression changes . Use metabolomics to detect alterations in carbon fixation pathways and energy metabolism . Conduct proteomics to identify changes in protein abundance or modification.

• Develop Rescue Experiments:

  • Issue: Determining causality of observed phenotypes.

  • Approach: Complement with wild-type gene. Create additional mutations in interacting proteins. Test suppressor mutations that may restore function. Introduce alternative electron transfer pathways.

By implementing this systematic troubleshooting framework, researchers can identify the molecular mechanisms underlying unexpected phenotypes in psbE mutants and distinguish between direct effects of the mutation and secondary adaptations of the photosynthetic machinery.

What strategies can help overcome challenges in purifying recombinant Synechococcus elongatus Cytochrome b559 alpha subunit for structural studies?

Purifying recombinant Cytochrome b559 alpha subunit from Synechococcus elongatus for structural studies presents numerous challenges due to its membrane-embedded nature, small size, and cofactor requirements. The following strategies can overcome these limitations:

• Optimize Expression Constructs:

  • Implement the pSyn_6 vector system which provides options for N-terminal and C-terminal affinity tags (His-tag, V5 epitope) to facilitate purification

  • Design constructs with TEV protease cleavage sites to remove tags after purification if needed for structural studies

  • Consider fusion partners that enhance solubility while maintaining structure

• Develop Specialized Solubilization Protocols:

  • Detergent screening: Systematically test multiple detergents (β-DDM, digitonin, LMNG) at various concentrations to identify optimal solubilization conditions

  • Detergent:protein ratio optimization: Determine precise ratios that efficiently extract the protein without destabilization

  • Gradient solubilization: Implement step-wise increases in detergent concentration to enhance selective extraction

• Multi-step Purification Strategy:

  • Begin with affinity chromatography using Ni-NTA for His-tagged constructs

  • Follow with ion exchange chromatography to remove contaminating proteins

  • Implement size exclusion chromatography as a final polishing step

  • Consider specialized techniques such as hydroxyapatite chromatography which has unique selectivity for heme-containing proteins

• Maintain Cofactor Association:

  • Supplement buffers with heme precursors during expression

  • Include stabilizing agents like glycerol (20-25%) in all buffers

  • Avoid strong reducing or oxidizing conditions that might alter heme coordination

  • Work under green light or dim light conditions to prevent photooxidative damage

• Complex Stabilization Approaches:

  • Co-express alpha and beta subunits to maintain the native heterodimeric structure

  • Consider purifying larger complexes (e.g., PSII subcomplexes) that maintain more native-like environments

  • Implement lipid supplementation during purification to maintain the lipid microenvironment

  • Use amphipols or nanodiscs for stabilization after initial purification

• Specialized Structural Biology Techniques:

  • For X-ray crystallography: Implement in situ proteolysis to remove flexible regions

  • For Cryo-EM: Consider gentler detergents (LMNG, GDN) that form smaller micelles

  • For NMR studies: Develop isotope labeling protocols specific for cyanobacterial expression

• Quality Control Throughout Purification:

  • Monitor spectral properties continuously (absorption peaks at 559 nm in reduced state)

  • Implement dynamic light scattering to assess monodispersity

  • Use thermal shift assays to evaluate stability in different buffer conditions

  • Employ native mass spectrometry to confirm complex integrity and stoichiometry

By implementing these specialized strategies, researchers can overcome the inherent challenges in purifying Cytochrome b559 alpha subunit while maintaining its structural integrity for high-resolution structural studies.

What analytical methods are most appropriate for evaluating changes in photosystem II function resulting from psbE modifications?

Comprehensive evaluation of photosystem II (PSII) function following psbE modifications requires a multi-parameter analytical approach. The following methods provide complementary insights into the functional consequences of Cytochrome b559 alpha subunit alterations:

• Oxygen Evolution Measurements:

  • Clark-type electrode analysis: Quantifies steady-state oxygen production rates under various light intensities

  • Flash-induced oxygen evolution: Measures the S-state transitions of the oxygen-evolving complex

  • Light saturation curves: Determines Pmax, quantum yield, and light compensation points

  • Application: Directly measures the primary function of PSII, revealing the efficiency of water-splitting and electron transport

• Chlorophyll Fluorescence Analysis:

  • Pulse-Amplitude Modulation (PAM) fluorometry: Measures Fv/Fm (maximum quantum yield), NPQ (non-photochemical quenching), and ETR (electron transport rate)

  • Fast fluorescence kinetics (OJIP transients): Reveals electron transfer steps within PSII

  • 77K fluorescence emission spectra: Distinguishes energy distribution between photosystems

  • Application: Non-invasive technique providing detailed information about PSII efficiency, energy dissipation mechanisms, and damage/repair dynamics

• Spectroscopic Analysis of Cytochrome b559:

  • Absorption difference spectroscopy: Quantifies high-potential and low-potential forms of Cytochrome b559

  • Electron paramagnetic resonance (EPR): Characterizes the heme environment and redox properties

  • Resonance Raman spectroscopy: Provides information about heme-protein interactions

  • Application: Directly assesses the molecular consequences of psbE modifications on the Cytochrome b559 structure and function

• Photoinhibition and Recovery Kinetics:

  • High-light challenge experiments: Measures the rate of PSII inactivation under photoinhibitory conditions

  • Recovery assays: Quantifies the kinetics of PSII repair following photodamage

  • Photoinhibition in the presence of protein synthesis inhibitors: Distinguishes between damage and repair processes

  • Application: Specifically evaluates the photoprotective role of Cytochrome b559, a function particularly sensitive to psbE modifications

• Thylakoid Electron Transport Measurements:

  • P700 oxidation-reduction kinetics: Assesses the electron flow from PSII to PSI

  • Artificial electron acceptor/donor assays: Isolates specific electron transport segments

  • Electrochromic shift measurements: Quantifies transmembrane electric field generation

  • Application: Provides information about integration of PSII function within the complete photosynthetic electron transport chain

• Protein Complex Analysis:

  • Blue Native-PAGE: Assesses PSII assembly, stability, and supercomplex formation

  • Two-dimensional gel electrophoresis: Identifies changes in PSII subunit composition

  • Pulse-chase labeling: Quantifies PSII turnover rates

  • Application: Evaluates whether psbE modifications affect PSII structural integrity and assembly

• Advanced Biophysical Techniques:

  • Thermoluminescence: Characterizes charge recombination events within PSII

  • Delayed fluorescence: Measures reverse electron flow within PSII

  • Circular dichroism: Detects structural changes in protein complexes

  • Application: Provides detailed biophysical information about altered energy transfer and electron transport mechanisms

• Transcriptomic and Proteomic Analysis:

  • RNA-Seq: Identifies compensatory gene expression changes

  • Quantitative proteomics: Measures alterations in protein abundance

  • Phosphoproteomics: Detects changes in regulatory post-translational modifications

  • Application: Reveals system-level adaptations to psbE modifications

This comprehensive analytical toolkit enables researchers to decipher the complex functional consequences of psbE modifications across multiple scales—from molecular alterations in Cytochrome b559 properties to system-level effects on photosynthetic performance.