Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the ...contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view.
A crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)−1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.
•Cooperative redox control of electron transfer mechanisms in proteins.•Continuous and strong illumination drives the photocycle without cyt bc1 complex.•The rate of photocycle depends on the size and redox poise of the periplasmic donor pool.•The natural electron donors have similar binding affinities to the tetraheme cytochrome and to the reaction center.•Ligands to both sides of the reaction center limit the rate of the photocycle.
In photosynthetic bacteria, the absorbed light drives the canonical cyclic electron transfer between the reaction center and the cytochrome
bc
1
complexes via the pools of mobile electron carriers. ...If kinetic or structural barriers hinder the participation of the
bc
1
complex in the cyclic flow of electrons, then the pools of mobile redox agents must supply the electrons for the multiple turnovers of the reaction center. These conditions were achieved by continuous high light excitation of intact cells of bacterial strains
Rba. sphaeroides
and
Rvx. gelatinosus
with depleted donor side cytochromes c
2
(
cycA
) and tetraheme cytochrome subunit (
pufC
), respectively. The gradual oxidation by ferricyanide further reduced the availability of electron donors to
pufC
. Electron transfer through the reaction center was tracked by absorption change and by induction and relaxation of the fluorescence of the bacteriochlorophyll dimer. The rate constants of the electron transfer (~ 3 × 10
3
s
‒1
) from the mobile donors of
Rvx. gelatinosus
bound either to the RC (
pufC
) or to the tetraheme subunit (wild type) were similar. The electrons transferred through the reaction center dimer were supplied entirely by the donor pool; their number amounted to about 5 in wild type
Rvx. gelatinosus
and decreased to 1 in
pufC
oxidized by ferricyanide. Fluorescence yield was measured as a function of the oxidized fraction of the dimer and its complex shape reveals the contribution of two competing processes: the migration of the excitation energy among the photosynthetic units and the availability of electron donors to the oxidized dimer. The experimental results were simulated and rationalized by a simple kinetic model of the two-electron cycling of the acceptor side combined with aperiodic one-electron redox function of the donor side.
Additive manufacturing technologies give many new application possibilities in our everyday life. Biomedical applications benefit a lot from 3D printing. In medical applications, several devices can ...be easily produced or prototyped with FFF/FDM technologies, but we must minimize the contamination risk. New materials regularly appear on the market, recently specified as antibacterial, resulting from compounded silver nanoparticles. Because scientifically accurate and practical information is not available, this way, we lack information regarding mechanical and thermal stability of the printed products. In addition, these parameters are essential in the setting and optimizing the 3D printers. In our recent study, we aimed to analyze PLA, PLA-HDT as well as PLA-Ag nanocomposite in the form of additive manufacturing filament, with DTA/TG. The results showed that these composites, based on their thermal characteristics, can be suitable for 3D print biomedical devices such as orthoses, casts, medical models and also surgical guides; therefore, their further examination should be important, regarding mechanical characteristics and their possible antibacterial effect.
Additive manufacturing technologies revolutionize many aspects of our everyday life. Biomedical applications benefit a lot from 3D printing. From surgical guides, patterns to custom-made splints and ...casts, several medical devices can be easily produced or prototyped with FFF/FDM technologies. New materials regularly appear on the market; therefore, the scientifically accurate and practical information is not available, and we are lack of information regarding mechanical and thermal stability of the printed products. In addition, these parameters are essential in setting and optimizing the 3D printers. In our study, we aimed to analyze two different, unique PLA/CaCO
3
composites in the form of additive manufacturing filament, with DTA. We tested the HDT form of PLA in pellet and filament form too. The results showed that these composites based on their thermal characteristics can be suitable for 3D print biomedical devices such as orthoses, casts, medical models and surgical guides too; therefore, their further examination should be important, regarding mechanical characteristics.
The development of photosynthetic membranes of intact cells of Rhodobacter sphaeroides was tracked by light-induced absorption spectroscopy and induction and relaxation of the bacteriochlorophyll ...fluorescence. Changes in membrane structure were induced by three methods: synchronization of cell growth, adjustment of different growth phases and transfer from aerobic to anaerobic conditions (greening) of the bacteria. While the production of the bacteriochlorophyll and carotenoid pigments and the activation of light harvesting and reaction center complexes showed cell-cycle independent and continuous increase with characteristic lag phases, the accumulation of phospholipids and membrane potential (electrochromism) exhibited stepwise increase controlled by cell division. Cells in the stationary phase of growth demonstrated closer packing and tighter energetic coupling of the photosynthetic units (PSU) than in their early logarithmic stage. The greening resulted in rapid (within 0–4 h) induction of BChl synthesis accompanied with a dominating role for the peripheral light harvesting system (up to LH2/LH1 ~2.5), significantly increased rate (~7·10⁴ s⁻¹) and yield (F ᵥ/F ₘₐₓ ~0.7) of photochemistry and modest (~2.5-fold) decrease of the rate of electron transfer (~1.5·10⁴ s⁻¹). The results are discussed in frame of a model of sequential assembly of the PSU with emphasis on crowding the LH2 complexes resulting in an increase of the connectivity and yield of light capture on the one hand and increase of hindrance to diffusion of mobile redox agents on the other hand.
The kinetics of bacteriochlorophyll fluorescence in intact cells of the purple nonsulfur bacterium
Rhodobacter sphaeroides
were measured under continuous and pulsed actinic laser diode (808 nm ...wavelength and maximum 2 W light power) illumination on the micro- and millisecond timescale. The fluorescence induction curve was interpreted in terms of a combination of photochemical and triplet fluorescence quenchers and was demonstrated to be a reflection of redox changes and electron carrier dynamics. By adjustment of the conditions of single and multiple turnovers of the reaction center, we obtained 11 ms
–1
and 120 μs
–1
for the rate constants of cytochrome
c
2
3+
detachment and cyclic electron flow, respectively. The effects of cytochrome
c
2
deletion and chemical treatments of the bacteria and the advantages of the fluorescence induction study on the operation of the electron transport chain
in vivo
were discussed.
Transition metal ions bind to the reaction center (RC) protein of the photosynthetic bacterium Rhodobacter sphaeroides and slow the light-induced electron and proton transfer to the secondary ...quinone, Q(B). We studied the properties of the metal ion-RC complex by measuring the pH dependence of the dissociation constant and the stoichiometry of proton release upon ligand formation. We investigated the mechanism of inhibition by measuring the stoichiometry and kinetics of flash-induced proton binding, the transfer of (first and second) electrons to Q(B), and the rate of steady-state turnover of the RC in the absence and presence of Cd(2+) and Ni(2+) on a wide pH range. The following results were obtained. (1) The complexation of transition metal ions Cd(2+) and Ni(2+) with the bacterial RC showed strong pH dependence. This observation was explained by different (pH-dependent) states of the metal-ligand cluster: the complex formation was strong when the ligand (Asp and His residues) was deprotonated and was much weaker if the ligand was partly (or fully) protonated. A direct consequence of the model was the pH-dependent proton release upon complexation. (2) The retardation of transfer of electrons and protons to Q(B) was also strongly pH-dependent. The effect was large in the neutral pH range and decreased toward the acidic and alkaline pH values. (3) Steady-state turnover measurements indicated that the rate of the second proton transfer was much less inhibited than that of the first one, which became the rate-limiting step in continuous turnover of the RC. (4) Sodium azide partly recovered the proton transfer rate. The effect is not due to removal of the bound metal ion by azide but probably by formation of a proton-transporting azide network similarly as water molecules may build up proton pathways. (5) We argue that the inhibition comes mainly from pK(a) shifts of key protonatable residues that control the proton transfer along the H-bond network to Q(B). The electrostatic interaction between the metal ion and these residues may result in acidic pK(a) shifts between 1.5 and 2.0 that account for the observed retardation of the electron and proton transfer.
The redox midpoint potential (
E
m) of Q
A, the primary quinone of bacterial reaction centers, is substantially modulated by the protein environment. Quite subtle mutations in the Q
A binding site, ...e.g., at residues M218, M252 and M265, cause significant increases in the equilibrium constant for electron transfer to Q
B, which indicate relative lowering of the
E
m of Q
A. However, reports of functional linkage between the Q
A and Q
B sites make it difficult to partition such effects between Q
A and Q
B from purely relative changes. We report here measurements on the yield of delayed fluorescence emission from the primary donor (P) accompanying the thermally activated charge recombination of P
+Q
A
− to form the excited singlet state of the primary donor, P*. The results show that for mutations of the Q
A site residues, Met
M218 and Ile
M265, essentially all the substantial thermodynamic effect is localized at Q
A, with no evidence for a significant effect of these residues on the properties of Q
B or the mutual influence (linkage) of Q
A and Q
B. We also report a significant lowering of the
E
m of Q
A by the native lipid, cardiolipin, which brings the
E
m in isolated reaction centers more in line with that seen in native membrane vesicles (chromatophores). Possible origins of this effect are discussed in the context of the Q
A binding site structure.
A minimal kinetic model of the photocycle, including both quinone (Q-6) reduction at the secondary quinone-binding site and (mammalian) cytochrome c oxidation at the cytochrome docking site of ...isolated reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides, was elaborated and tested by cytochrome photooxidation under strong continuous illumination. The typical rate of photochemical excitation by a laser diode at 810 nm was 2.200 s-1, and the rates of stationary turnover of the reaction center (one-half of that of cytochrome photooxidation) were 600 +/- 70 s-1 at pH 6 and 400 +/- 50 s-1 at pH 8. The rate of turnover showed strong pH dependence, indicating the contribution of different rate-limiting processes. The kinetic limitation of the photocycle was attributed to the turnover of the cytochrome c binding site (pH < 6), light intensity and quinone/quinol exchange (6
< pH < 8), and proton-coupled second electron transfer in the quinone acceptor complex (pH > 8). The analysis of the double-reciprocal plot of the rate of turnover versus light intensity has proved useful in determining the light-independent (maximum) turnover rate of the reaction center (445 +/- 50 s-1 at pH 7.8).
Kinetics and stoichiometry of proton binding/unbinding induced by intense (1 W cm-2) and continuous illumination were measured in the isolated reaction center (RC) protein from photosynthetic purple ...bacterium Rhodobacter sphaeroides in the absence of an external electron donor. At high ionic strength (100 mM), large proton release (≈6 H+ per RC) was observed at pH 6 and substoichiometric H+-ion binding (≈0.3 H+ per RC) at pH 8. These observations together with optical spectroscopy on the oxidized dimer indicate that, at room temperature, two distinct conformations of the RC can be obtained depending on the pH, E h, and illumination. Acidic pH, a large redox gap between the actual E h of the solution and the midpoint potential of the acceptor quinone, and strong illumination favor the conversion of the RC from the dark-adapted state to the light-adapted state. These conformations differ greatly in the rates of primary photochemistry, the reoxidation of semiquinone and the rereduction of the oxidized dimer, and the protonation states of the amino acids of the protein. Whereas substoichiometric proton unbinding is observed in the P+Q redox state of the protein in the dark-adapted conformation, much larger H+-ion release is detected in the light-adapted conformation. From the pH dependence of the key processes in the conformational change and reoxidation of semiquinone, we concluded that they are controlled by protonatable groups available in the protein. A simple phenomenological model is presented that relates the rates and equilibrium constants of the electron transfer reactions and the conformational change of the RC.
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