Exploring the Function of Cytochrome c6A

The electron transfer protein cytochrome c6 (c6) of photosynthesis was believed to have been lost in plants until a ubiquitous and highly conserved homologue, named cytochrome c6A (c6A), was identified. Soon after its discovery, c6A was shown to be unable to replace c6 (or plastocyanin) functionally in the photosynthetic electron transfer chain (PETC), despite their significant structural homology. Since then only few studies have revealed additional insights about c6A. Thus its function remains unknown, although the high sequence conservation of c6A across the green lineage points towards an important role. In this study, the function of c6A in the green alga Chlamydomonas reinhardtii was explored. A wide range of microbiological, biochemical, and biophysical techniques was employed to compare c6A knock-out and overexpression strains to the wild-type background. Exposure to a harsh fluctuating light regime, named DISCO Light, in photomixotrophy was found to lead to a growth penalty in the absence of c6A. No other significant growth phenotypes could be observed in continuous light, continuous darkness, or milder fluctuating light regimes in photoautotrophic or photomixotrophic conditions. The light dependency of the growth phenotype and homology of c6A to c6 prompted in-depth analyses of the PETC using absorbance and fluorescence spectrometry. In the absence of c6A, the light harvesting balance between PSI and PSII was shifted in favour of PSII and the PQ pool was more reduced, even in light conditions that did not cause a growth phenotype. While state transitions were at least in principle functional, the shift in light harvesting towards PSII was still present in conditions that would normally lead to state 1 or state 2. The more reduced PQ pool was found to have a prominent effect on the redox kinetics of b hemes in the cytochrome b6f complex in response to light, while the Q cycle appeared functional. In DISCO Light, the c6A-dependent phenotype of an altered light harvesting balance was amplified, the photochemical activity of PSII was lower without c6A, and elevated NPQ levels suggested increased stress levels in the absence of c6A. These insights explain the observed growth penalty which could be caused by the altered light harvesting balance, leading to a more reduced PQ pool, and ultimately increased damage through photooxidative stress in the absence of c6A. To investigate further a possible mechanism of action, the localisation and interactions of c6A were studied. Using subcellular fractionation and immunofluorescence microscopy c6A was found in the thylakoid lumen. Co-immunoprecipitation studies did not reveal any stable complexes, consistent with a function for c6A through transient interactions. Examination of the effect of c6A on complexes of the thylakoid membrane showed a putative LHC-less PSI complex that was more prominent in the absence of c6A. Western blot analysis indicated that the abundances of PSI and PSII core proteins were unlikely to account for the increased presence of this PSI complex, indicating that changes to the light harvesting antennae are more likely underlying this observation. Reduced abundance of PTOX2, a ‘safety valve’ for excess electrons in the PETC, was detected in the absence of c6A, potentially contributing to the observed difference in PQ pool redox state. RNAseq revealed c6A-dependent changes in transcript levels of genes associated with circadian rhythm, chloroplast thylakoid components including light-harvesting complex subunits, and thiol-based redox regulation, even in conditions that did not cause a growth phenotype. In addition, in DISCO Light, a decreased expression of oxidative stress response genes in the c6A knock-out compared to wild-type was found, which could further contribute to the observed growth phenotype. Correspondingly, a transient increase in abundance of the stress-related NPQ effector LHCSR3 and the prominent emergence of PsbA breakdown products in the absence of c6A in DISCO Light also indicated increased oxidative stress. Previous hypotheses for the function of c6A proposed that it is a replacement for plastocyanin, a safety valve for transfer of excess electrons, a high light signalling protein or, via its conserved disulfide bridge, a redox-regulator in the thylakoid lumen. The latter appeared the most likely in the light of the results of this study and the model for this was developed further. It is proposed that c6A influences photosynthetic performance via fine-tuning the redox state of thiol-based redox regulation targets in the lumen, like the state transition kinase STT7 and the PSII subunit PSBO. It is further suggested that the ability of c6A to transfer electrons via its bound heme and the disulphide bridge to multiple electron acceptors provides an additional degree of flexibility, important to maintain the complex luminal redox homeostasis, especially under stressful environmental conditions.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2024

Author dc:creator

• Kosmützky, Darius

Advisor dc:contributor.advisor

• Howe, Christopher

• Chlamydomonas reinhardtii
• Cytochrome c6A
• DISCO Light
• Electron transfer protein
• Fluctuating light
• Photosynthesis
• Photosynthetic electron transfer chain

Licence

• https://www.repository.cam.ac.uk/bitstreams/0b0a462f-0e9a-40a9-9935-f49a0a1f91de/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.116162

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/380596