Cytochromes C Research Articles

Hydrogen gas as a central on-off functional switch of reversible metabolic arrest – New perspectives for biotechnological applications

Metabolism is the sum of all chemical reactions that sustain life. There is an ongoing effort to control metabolic rate, which correlates with the maximum lifespan potential and constitutes one of the oldest scientific questions. Herein, we report on the complete reversible arrest of cellular metabolism and cell growth in a series of organisms, from microalgae to yeast upon exposure to a 100 % hydrogen atmosphere. We also report a tolerance of the microalgae under these conditions against extreme stress conditions, like high salt concentrations. The addition of oxygen or air almost completely restores the metabolic rate and cell growth. Molecular dynamics simulations are employed to decipher this phenomenon at atomic scale. Various proteins, including photosynthetic and respiratory complexes (LHCII, cytochrome c5) are probed in the interaction with hydrogen. Exposure to hydrogen, as opposed to oxygen, decreases the fluctuations of protein residues indicating thermostability. According to the above mechanism, an absolute hydrogen atmosphere can preserve biological products (e.g. fruits) for a long time without consuming any energy. By combining biological, chemical and computational methods, in this research we provide the basis for future innovative studies and advances in the field of biotechnology.

Deletion of the cytochrome bc complex from Heliobacterium modesticaldum results in viable but non-phototrophic cells.

The heliobacteria, a family of anoxygenic phototrophs, possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria can also grow chemotrophically via pyruvate metabolism in the dark. In the heliobacteria, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for the mobile cytochrome c553 other than the photochemical reaction center. We have, therefore, hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. We used a two-step method for CRISPR-based genome editing in Heliobacterium modesticaldum to delete the genes encoding the four major subunits of the cytochrome bc complex. Genotypic analysis verified the deletion of the petCBDA gene cluster encoding the catalytic components of the complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was over 100 times slower in the ∆petCBDA mutant compared to the wild-type. Steady-state levels of oxidized P800 (the primary donor of the photochemical reaction center) were much higher in the ∆petCBDA mutant at every light level, consistent with a limitation in electron flow to the reaction center. The ∆petCBDA mutant was unable to grow phototrophically on acetate plus CO2 but could grow chemotrophically on pyruvate as a carbon source similar to the wild-type strain in the dark. The mutants could be complemented by reintroduction of the petCBDA gene cluster on a plasmid expressed from the clostridial eno promoter.

In vivo electron donation from plastocyanin and cytochrome c6 to PSI in Synechocystis sp. PCC6803

Many cyanobacteria species can use both plastocyanin and cytochrome c6 as lumenal electron carriers to shuttle electrons from the cytochrome b6f to either photosystem I or the respiratory cytochrome c oxidase. In Synechocystis sp. PCC6803 placed in darkness, about 60% of the active PSI centres are bound to a reduced electron donor which is responsible for the fast re-reduction of P700in vivo after a single charge separation. Here, we show that both cytochrome c6 and plastocyanin can bind to PSI in the dark and participate to the fast phase of P700 reduction, but the fraction of pre-bound PSI is smaller in the case of cytochrome c6 than with plastocyanin. Because of the inter-connection of respiration and photosynthesis in cyanobacteria, the inhibition of the cytochrome c oxidase results in the over-reduction of the photosynthetic electron transfer chain in the dark that translates into a lag in the kinetics of P700 oxidation at the onset of light. We show that this is true both with plastocyanin and cytochrome c6, indicating that the partitioning of electron transport between respiration and photosynthesis is regulated in the same way independently of which of the two lumenal electron carriers is present, although the mechanisms of such regulation are yet to be understood.

A graph theoretic model to understand the behavioral difference of PPCA among its paralogs towards recognition of DXCA

Among all the proteins of Periplasmic C type Cytochrome family obtained from cytochrome C7 found in Geobacter sulfurreducens, only the Periplasmic C type Cytochrome A (PPCA) protein can recognize the deoxycholate (DXCA), while its other paralogs do not, as observed from the crystal structures. Though some existing works have used graph-theoretic approaches to realize the 3-D structural properties of proteins, its usage in the rationalisation of the physiochemical behavior of proteins has been very limited. To understand the driving force towards the recognition of DXCA exclusively by PPCA among its paralogs, in this work, we propose two graph theoretic models based on the combinatorial properties, namely, base-pair-type and impact, of the nucleotide bases and the amino acid residues, respectively. Combinatorial analysis of the binding sequences using the proposed base-pair type based graph theoretic model reveals the differential behaviour of PPCA among its other paralogs. Further, to investigate the underlying chemical phenomenon, another graph theoretic model has been developed based on impact. Analysis of the results obtained from impact-based model clearly indicates towards the helix formation of PPCA which is essential for the recognition of DXCA, making PPCA a completely different entity from its paralogs.

Very Low-Frequency Broadband Electron Paramagnetic Resonance Spectroscopy of Metalloproteins.

A previously developed spectrometer for broadband electron paramagnetic resonance (EPR) spectroscopy of dilute randomly oriented systems has been considerably modified to extend the frequency reach down to the hundred MHz range and to boost concentration sensitivity by 1 to 2 orders of magnitude. The instrument is now suitable for the study of biological systems in particular metalloproteins. As a proof of concept, examples from the class of low-spin ferric hemoproteins are studied in terms of frequency-dependent changes in their EPR spectra. Mono-heme cytochrome c EPR is determined by g-strain over a wide frequency range, whereas a combination of unresolved ligand hyperfine interaction and concentration-dependent intermolecular dipolar interaction becomes dominant at very low frequencies. In the four heme containing cytochrome c3, g-strain combines with intramolecular dipolar interaction over the full-studied frequency range of 0.23–12.0 GHz. It is concluded that the point-dipole approach is inappropriate to describe magnetic interactions between low-spin ferric heme systems and that a body of literature on redox interactions in multi-heme proteins will be affected by this conclusion.

Enhancement of direct electron transfer in graphene bioelectrodes containing novel cytochrome c553 variants with optimized heme orientation

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochromec553(cytc553)peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cytc553holoprotein and thesurface anchoring C-terminal His6-tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c553 leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers.

New Insights into the Evolution of the Electron Transfer from Cytochrome f to Photosystem I in the Green and Red Branches of Photosynthetic Eukaryotes

In cyanobacteria and most green algae of the eukaryotic green lineage, the copper-protein plastocyanin (Pc) alternatively replaces the heme-protein cytochrome c6 (Cc6) as the soluble electron carrier from cytochrome f (Cf) to photosystem I (PSI). The functional and structural equivalence of ‘green’ Pc and Cc6 has been well established, representing an example of convergent evolution of two unrelated proteins. However, plants only produce Pc, despite having evolved from green algae. On the other hand, Cc6 is the only soluble donor available in most species of the red lineage of photosynthetic organisms, which includes, among others, red algae and diatoms. Interestingly, Pc genes have been identified in oceanic diatoms, probably acquired by horizontal gene transfer from green algae. However, the mechanisms that regulate the expression of a functional Pc in diatoms are still unclear. In the green eukaryotic lineage, the transfer of electrons from Cf to PSI has been characterized in depth. The conclusion is that in the green lineage, this process involves strong electrostatic interactions between partners, which ensure a high affinity and an efficient electron transfer (ET) at the cost of limiting the turnover of the process. In the red lineage, recent kinetic and structural modeling data suggest a different strategy, based on weaker electrostatic interactions between partners, with lower affinity and less efficient ET, but favoring instead the protein exchange and the turnover of the process. Finally, in diatoms the interaction of the acquired green-type Pc with both Cf and PSI may not yet be optimized.

Complete genome sequence of Shewanella marisflavi ECSMB14101, a red pigment synthesizing bacterium isolated from the East China Sea.

Bacteria of the genus Shewanella have been studied for their versatile electron-accepting abilities, particularly for extracellular electron transfer via minerals. Shewanella marisflavi ECSMB14101 was isolated from naturally formed biofilms in the East China Sea. The genome of S. marisflavi ECSMB14101 encodes 3891 genes with a total size of 4,343,492bp in one chromosome. Its GC content is 49.89%. S. marisflavi ECSMB14101 is able to synthesize a red pigment, which may be achieved through Cytochrome c3 and electron transfer to reduce Fe(III) oxide. The genomic data presented here could provide fundamental insights to better understand the physiological characteristics of S. marisflavi, the ecological significance of red pigment synthesis, and its inductive effects on the settlement of marine invertebrate larvae.

A protease-mediated mechanism regulates the cytochrome c6/plastocyanin switch in Synechocystis sp. PCC 6803

After the Great Oxidation Event (GOE), iron availability was greatly decreased, and photosynthetic organisms evolved several alternative proteins and mechanisms. One of these proteins, plastocyanin, is a type I blue-copper protein that can replace cytochrome c6 as a soluble electron carrier between cytochrome b6f and photosystem I. In most cyanobacteria, expression of these two alternative proteins is regulated by copper availability, but the regulatory system remains unknown. Herein, we provide evidence that the regulatory system is composed of a BlaI/CopY-family transcription factor (PetR) and a BlaR-membrane protease (PetP). PetR represses petE (plastocyanin) expression and activates petJ (cytochrome c6), while PetP controls PetR levels in vivo. Using whole-cell extracts, we demonstrated that PetR degradation requires both PetP and copper. Transcriptomic analysis revealed that the PetRP system regulates only four genes (petE, petJ, slr0601, and slr0602), highlighting its specificity. Furthermore, the presence of petE and petRP in early branching cyanobacteria indicates that acquisition of these genes could represent an early adaptation to decreased iron bioavailability following the GOE.

Biotransformation mechanism of Vibrio diabolicus to sulfamethoxazole at transcriptional level

Sulfamethoxazole (SMX) has attracted much attention due to its high probability of detection in the environment. Marine bacteria Vibrio diabolicus strain L2–2 has been proven to be able to transform SMX. In this study, the potential resistance and biotransformation mechanism of strain L2–2 to SMX, and key genes responses to SMX at environmental concentrations were researched. KEGG pathways were enriched by down-regulated genes including degradation of L-Leucine, L-Isoleucine, and fatty acid metabolism. Resistance mechanism could be concluded as the enhancement of membrane transport, antioxidation, response regulator, repair proteins, and ribosome protection. Biotransformation genes might involve in arylamine N-acetyltransferases (nat), cytochrome c553 (cyc-553) and acyl-CoA synthetase (acs). At the environmental concentration of SMX (0.1–10 μg/L), nat was not be activated, which meant the acetylation of SMX might not occur in the environment; however, cyc-553 was up-regulated under SMX stress of 1 μg/L, which indicated the hydroxylation of SMX could occur in the environment. Besides, the membrane transport and antioxidation of strain L2–2 could be activated under SMX stress of 10 μg/L. The results provided a better understanding of resistance and biotransformation of bacteria to SMX and would support related researches about the impacts of environmental antibiotics.

Development of a universal conductive platform for anchoring photo- and electroactive proteins using organometallic terpyridine molecular wires.

The construction of an efficient conductive interface between electrodes and electroactive proteins is a major challenge in the biosensor and bioelectrochemistry fields to achieve the desired nanodevice performance. Concomitantly, metallo-organic terpyridine wires have been extensively studied for their great ability to mediate electron transfer over a long-range distance. In this study, we report a novel stepwise bottom-up approach for assembling bioelectrodes based on a genetically modified model electroactive protein, cytochrome c553 (cyt c553) and an organometallic terpyridine (TPY) molecular wire self-assembled monolayer (SAM). Efficient anchoring of the TPY derivative (TPY-PO(OH)2) onto the ITO surface was achieved by optimising solvent composition. Uniform surface coverage with the electroactive protein was achieved by binding the cyt c553 molecules via the C-terminal His6-tag to the modified TPY macromolecules containing Earth abundant metallic redox centres. Photoelectrochemical characterisation demonstrates the crucial importance of the metal redox centre for the determination of the desired electron transfer properties between cyt and the ITO electrode. Even without the cyt protein, the ITO-TPY nanosystem reported here generates photocurrents whose densities are 2-fold higher that those reported earlier for ITO electrodes functionalised with the photoactive proteins such as photosystem I in the presence of an external mediator, and 30-fold higher than that of the pristine ITO. The universal chemical platform for anchoring and nanostructuring of (photo)electroactive proteins reported in this study provides a major advancement for the construction of efficient (bio)molecular systems requiring a high degree of precise supramolecular organisation as well as efficient charge transfer between (photo)redox-active molecular components and various types of electrode materials.

Contributions to cytochrome c inner- and outer-sphere reorganization energy.

Cytochromes c are small water-soluble proteins that catalyze electron transfer in metabolism and energy conversion processes. Hydrogenobacter thermophilus cytochrome c552 presents a curious case in displaying fluxionality of its heme axial methionine ligand; this behavior is altered by single point mutation of the Q64 residue to N64 or V64, which fixes the ligand in a single configuration. The reorganization energy (λ) of these cytochrome c552 variants is experimentally determined using a combination of rotating disc electrochemistry, chronoamperometry and cyclic voltammetry. The differences between the λ determined from these complementary techniques helps to deconvolute the contribution of the active site and its immediate environment to the overall λ (λTotal). The experimentally determined λ values in conjunction with DFT calculations indicate that the differences in λ among the protein variants are mainly due to the differences in contributions from the protein environment and not just inner-sphere λ. DFT calculations indicate that the position of residue 64, responsible for the orientation of the axial methionine, determines the geometric relaxation of the redox active molecular orbital (RAMO). The orientation of the RAMO with respect to the heme is key to determining electron transfer coupling (HAB) which results in higher ET rates in the wild-type protein relative to the Q64V mutant despite a 150 mV higher λTotal in the former.

Increase of complex I and reduction of complex II mitochondrial activity are possible adaptive effects provoked by fluoride exposure

Fluoride (F) can induce changes in the expression of several liver proteins, most of them localized in the mitochondria and its effect is dose- and time-dependent. This study analyzed the effect of distinct F concentrations and exposure periods on the mitochondrial activity of complex I-III and II-III in the liver. Thirty-six 21-day-old male Wistar rats were divided into 2 groups (n = 18) according to the duration of the treatment (20 or 60 days). They were subdivided into 3 subgroups (n = 6) according to the concentration of F (0 mg/L, 15 mg/L or 50 mg/L). After the experimental periods, the animals were anesthetized, liver mitochondria were isolated and stored for activity analyses. The determination of complexes II-III and I-III was based on the reduction of cytochrome c3+ to cytochrome c2+ performed spectrophotometrically. Bioinformatics analyses were performed using data from a previous study (Pereira et al., 2018). The mitochondrial complex I-III was significantly activated in the groups treated with 50 mgF/L for 20 days and 15 mgF/L for 60 days. The complex II-III was significantly reduced in the group treated with the higher F dose for 60 days. The networks indicated more changes in mitochondrial proteins in the group treated with the higher dose for 20 days; the reduction is probably linked to the activation of the complex I-III. The reduction in the complex II-III upon exposure to the higher F dose in the long term might be part of an adaptative mechanism of the body to counteract the deleterious effects of this ion on the energy metabolism.

The heterologous expression of a plastocyanin in the diatom Phaeodactylum tricornutum improves cell growth under iron-deficient conditions.

We have investigated if the heterologous expression of a functional green alga plastocyanin in the diatom Phaeodactylum tricornutum can improve photosynthetic activity and cell growth. Previous in vitro assays showed that a single-mutant of the plastocyanin from the green algae Chlamydomonas reinhardtii is effective in reducing P. tricornutum photosystem I. In this study, in vivo assays with P. tricornutum strains expressing this plastocyanin indicate that even the relatively low intracellular concentrations of holo-plastocyanin detected (≈4 μM) are enough to promote an increased growth (up to 60%) under iron-deficient conditions as compared with the WT strain, measured as higher cell densities, content in pigments and active photosystem I, global photosynthetic rates per cell, and even cell volume. In addition, the presence of plastocyanin as an additional photosynthetic electron carrier seems to decrease the over-reduction of the plastoquinone pool. Consequently, it promotes an improvement in the maximum quantum yield of both photosystem II and I, together with a decrease in the acceptor side photoinhibition of photosystem II-also associated to a reduced oxidative stress-a decrease in the peroxidation of membrane lipids in the choroplast, and a lower degree of limitation on the donor side of photosystem I. Thus the heterologous plastocyanin appears to act as a functional electron carrier, alternative to the native cytochrome c6 , under iron-limiting conditions.

A Model of Aerobic and Anaerobic Metabolism of Hydrogen in the Extremophile Acidithiobacillus ferrooxidans

Hydrogen can serve as an electron donor for chemolithotrophic acidophiles, especially in the deep terrestrial subsurface and geothermal ecosystems. Nevertheless, the current knowledge of hydrogen utilization by mesophilic acidophiles is minimal. A multi-omics analysis was applied on Acidithiobacillus ferrooxidans growing on hydrogen, and a respiratory model was proposed. In the model, [NiFe] hydrogenases oxidize hydrogen to two protons and two electrons. The electrons are used to reduce membrane-soluble ubiquinone to ubiquinol. Genetically associated iron-sulfur proteins mediate electron relay from the hydrogenases to the ubiquinone pool. Under aerobic conditions, reduced ubiquinol transfers electrons to either cytochrome aa3 oxidase via cytochrome bc1 complex and cytochrome c4 or the alternate directly to cytochrome bd oxidase, resulting in proton efflux and reduction of oxygen. Under anaerobic conditions, reduced ubiquinol transfers electrons to outer membrane cytochrome c (ferrireductase) via cytochrome bc1 complex and a cascade of electron transporters (cytochrome c4, cytochrome c552, rusticyanin, and high potential iron-sulfur protein), resulting in proton efflux and reduction of ferric iron. The proton gradient generated by hydrogen oxidation maintains the membrane potential and allows the generation of ATP and NADH. These results further clarify the role of extremophiles in biogeochemical processes and their impact on the composition of the deep terrestrial subsurface.

Insight into Electron Transfer from a Redox Polymer to a Photoactive Protein.

Biohybrid photoelectrochemical systems in photovoltaic or biosensor applications have gained considerable attention in recent years. While the photoactive proteins engaged in such systems usually maintain an internal charge separation quantum yield of nearly 100%, the subsequent steps of electron and hole transfer beyond the protein often limit the overall system efficiency and their kinetics remain largely uncharacterized. To reveal the dynamics of one of such charge-transfer reactions, we report on the reduction of Rhodobacter sphaeroides reaction centers (RCs) by Os-complex-modified redox polymers (P-Os) characterized using transient absorption spectroscopy. RCs and P-Os were mixed in buffered solution in different molar ratios in the presence of a water-soluble quinone as an electron acceptor. Electron transfer from P-Os to the photoexcited RCs could be described by a three-exponential function, the fastest lifetime of which was on the order of a few microseconds, which is a few orders of magnitude faster than the internal charge recombination of RCs with fully separated charge. This was similar to the lifetime for the reduction of RCs by their natural electron donor, cytochrome c2. The rate of electron donation increased with increasing ratio of polymer to protein concentrations. It is proposed that P-Os and RCs engage in electrostatic interactions to form complexes, the sizes of which depend on the polymer-to-protein ratio. Our findings throw light on the processes within hydrogel-based biophotovoltaic devices and will inform the future design of materials optimally suited for this application.

Heterologous production and functional characterization of Bradyrhizobium japonicum copper-containing nitrite reductase and its physiological redox partner cytochrome c550.

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions.

Insights into growth kinetics and roles of enzymes of Krebs' cycle and sulfur oxidation during exochemolithoheterotrophic growth of Achromobacter aegrifaciens NCCB 38021 on succinate with thiosulfate as the auxiliary electron donor.

Achromobacter aegrifaciens NCCB 38021 was grown heterotrophically on succinate versus exochemolithoheterotrophically on succinate with thiosulfate as auxiliary electron donor. In batch culture, no significant differences in specific molar growth yield or specific growth rate were found for the two growth conditions, but in continuous culture in the succinate-limited chemostat, the maximum specific growth yield coefficient increased by 23.3% with thiosulfate present, consistent with previous studies of endo- and exochemolithoheterotrophs and thermodynamic predictions. Thiosulfate oxidation was coupled to respiration at cytochrome c551, and thiosulfate-dependent ATP biosynthesis occurred. Specific activities of cytochrome c-linked thiosulfate dehydrogenase (E.C. 1.8.2.2) and two other enzymes of sulfur metabolism were significantly higher in exochemolithoheterotrophically grown cell extracts, while those of succinyl-transferring 2-oxoglutarate dehydrogenase (E.C. 1.2.4.2), fumarate hydratase (E.C. 4.2.1.2) and malate dehydrogenase (NAD+, E.C. 1.1.1.37) were significantly lower-presumably owing to less need to generate reducing equivalents during Krebs' cycle, since they could be produced from thiosulfate oxidation.

Crystal structures of native cytochrome c6 from Thermosynechococcus elongatus in two different space groups and implications for its oligomerization.

Native cytochrome c6 was purified from an extract of strain BP-1 of the thermophilic cyanobacterium Thermosynechococcus elongatus. The protein was crystallized, and with only slight modifications of the buffer and vapour-diffusion conditions two different space groups were observed, namely H3 and C2. Both crystal structures were solved; they contained three and six molecules per asymmetric unit and were refined to 1.7 and 2.25 Å resolution, respectively. To date, the structure of native cytochrome c6 from T. elongatus has only been reported as a monomer using NMR spectroscopy, i.e. without addressing putative oligomerization, and related structures have only previously been solved using X-ray crystallography after recombinant gene overexpression in Escherichia coli. The reported space groups of related cyanobacterial cytochrome c6 structures differ from those reported here. Interestingly, the protein-protein interfaces that were observed utilizing X-ray crystallography could also explain homo-oligomerization in solution; specifically, trimerization is indicated by infra-red dynamic light scattering and blue native gel electrophoresis in solution. Trimers were also detected by mass spectrometry. Furthermore, there is an indication of post-translational methylation in the crystal structure. Additionally, the possibility of modifying the crystal size and the redox activity in the context of photosynthesis is shaping the investigated cytochrome as a highly suitable model protein for advanced serial crystallography at highly brilliant X-ray free-electron laser sources.

Correlated clusters of closed reaction centers during induction of intact cells of photosynthetic bacteria

Antenna systems serve to absorb light and to transmit excitation energy to the reaction center (RC) in photosynthetic organisms. As the emitted (bacterio)chlorophyll fluorescence competes with the photochemical utilization of the excitation, the measured fluorescence yield is informed by the migration of the excitation in the antenna. In this work, the fluorescence yield concomitant with the oxidized dimer (P+) of the RC were measured during light excitation (induction) and relaxation (in the dark) for whole cells of photosynthetic bacterium Rhodobacter sphaeroides lacking cytochrome c2 as natural electron donor to P+ (mutant cycA). The relationship between the fluorescence yield and P+ (fraction of closed RC) showed deviations from the standard Joliot–Lavergne–Trissl model: (1) the hyperbola is not symmetric and (2) exhibits hysteresis. These phenomena originate from the difference between the delays of fluorescence relative to P+ kinetics during induction and relaxation, and in structural terms from the non-random distribution of the closed RCs during induction. The experimental findings are supported by Monte Carlo simulations and by results from statistical physics based on random walk approximations of the excitation in the antenna. The applied mathematical treatment demonstrates the generalization of the standard theory and sets the stage for a more adequate description of the long-debated kinetics of fluorescence and of the delicate control and balance between efficient light harvest and photoprotection in photosynthetic organisms.