bacteriochlorophylls and quinone

Insight into control of proton transfer, a crucial attribute of cellular functions, can be gained from investigations of bacterial reaction centers. While the uptake of protons associated with the reduction of the quinone is well characterized, the release of protons associated with the oxidized bacteriochlorophyll dimer has been poorly understood. Optical spectroscopy and proton release/uptake measurements were used to examine the proton release characteristics of twelve mutant reaction centers, each containing a change in an amino acid residue near the bacteriochlorophyll dimer. The mutant reaction centers had optical spectra similar to wild-type and were capable of transferring electrons to the quinones after light excitation of the bacteriochlorophyll dimer. They exhibited a large range in the extent of proton release and in the slow recovery of the optical signal for the oxidized dimer upon continuous illumination. Key roles were indicated for six amino acid residues, Thr L130, Asp L155, Ser L244, Arg M164, Ser M190, and His M193. Analysis of the results points to a hydrogen-bond network that contains these residues, with several additional residues and bound water molecules, forming a proton transfer pathway. In addition to proton transfer, the properties of the pathway are proposed to be responsible for the very slow charge recombination kinetics observed after continuous illumination. The characteristics of this pathway are compared to proton transfer pathways near the secondary quinone as well as those found in photosystem II and cytochrome c oxidase.

Topics: Amino Acids; Bacteriochlorophylls; Electron Transport; Hydrogen-Ion Concentration; Mutagenesis, Site-Directed; Oxidation-Reduction; Photosynthetic Reaction Center Complex Proteins; Protons; Rhodobacter sphaeroides

The reaction centre light-harvesting 1 (RC-LH1) complex is the core functional component of bacterial photosynthesis. We determined the cryo-electron microscopy (cryo-EM) structure of the RC-LH1 complex from Rhodospirillum rubrum at 2.5 Å resolution, which reveals a unique monomeric bacteriochlorophyll with a phospholipid ligand in the gap between the RC and LH1 complexes. The LH1 complex comprises a circular array of 16 αβ-polypeptide subunits that completely surrounds the RC, with a preferential binding site for a quinone, designated QP, on the inner face of the encircling LH1 complex. Quinols, initially generated at the RC QB site, are proposed to transiently occupy the QP site prior to traversing the LH1 barrier and diffusing to the cytochrome bc1 complex. Thus, the QP site, which is analogous to other such sites in recent cryo-EM structures of RC-LH1 complexes, likely reflects a general mechanism for exporting quinols from the RC-LH1 complex.

Topics: Bacterial Proteins; Bacteriochlorophylls; Benzoquinones; Binding Sites; Cryoelectron Microscopy; Crystallization; Electron Transport Complex III; Hydrogen Bonding; Hydroquinones; Ligands; Light-Harvesting Protein Complexes; Phospholipids; Protein Conformation, alpha-Helical; Rhodospirillum rubrum

The light-harvesting 1-reaction centre (LH1-RC) complex is a key functional component of bacterial photosynthesis. Here we present a 2.9 Å resolution cryo-electron microscopy structure of the bacteriochlorophyll b-based LH1-RC complex from Blastochloris viridis that reveals the structural basis for absorption of infrared light and the molecular mechanism of quinone migration across the LH1 complex. The triple-ring LH1 complex comprises a circular array of 17 β-polypeptides sandwiched between 17 α- and 16 γ-polypeptides. Tight packing of the γ-apoproteins between β-polypeptides collectively interlocks and stabilizes the LH1 structure; this, together with the short Mg-Mg distances of bacteriochlorophyll b pairs, contributes to the large redshift of bacteriochlorophyll b absorption. The 'missing' 17th γ-polypeptide creates a pore in the LH1 ring, and an adjacent binding pocket provides a folding template for a quinone, Q

Topics: Apoproteins; Bacterial Proteins; Bacteriochlorophylls; Benzoquinones; Binding Sites; Cryoelectron Microscopy; Hyphomicrobiaceae; Light-Harvesting Protein Complexes; Magnesium; Models, Molecular; Photosynthesis; Protein Conformation; Protein Stability

Reaction center-light harvesting 1 (RC-LH1) complexes are the fundamental units of bacterial photosynthesis, which use solar energy to power the reduction of quinone to quinol prior to the formation of the proton gradient that drives ATP synthesis. The dimeric RC-LH1-PufX complex of Rhodobacter sphaeroides is composed of 64 polypeptides and 128 cofactors, including 56 LH1 bacteriochlorophyll a (BChl a) molecules that surround and donate energy to the two RCs. The 3D structure was determined to 8 Å by X-ray crystallography, and a model was built with constraints provided by electron microscopy (EM), nuclear magnetic resonance (NMR), mass spectrometry (MS), and site-directed mutagenesis. Each half of the dimer complex consists of a RC surrounded by an array of 14 LH1 αβ subunits, with two BChls sandwiched between each αβ pair of transmembrane helices. The N- and C-terminal extrinsic domains of PufX promote dimerization by interacting with the corresponding domains of an LH1 β polypeptide from the other half of the RC-LH1-PufX complex. Close contacts between PufX, an LH1 αβ subunit, and the cytoplasmic domain of the RC-H subunit prevent the LH1 complex from encircling the RC and create a channel connecting the RC QB site to an opening in the LH1 ring, allowing Q/QH₂ exchange with the external quinone pool. We also identified a channel that connects the two halves of the dimer, potentially forming a long-range pathway for quinone migration along rows of RC-LH1-PufX complexes in the membrane. The structure of the RC-LH1-PufX complex explains the crucial role played by PufX in dimer formation, and it shows how quinone traffic traverses the LH1 complex as it shuttles between the RC and the cytochrome bc₁ complex.

Topics: Bacterial Proteins; Bacteriochlorophyll A; Benzoquinones; Carotenoids; Light-Harvesting Protein Complexes; Mass Spectrometry; Models, Molecular; Oxidation-Reduction; Protein Conformation; Protein Interaction Domains and Motifs; Protein Multimerization; Protein Structure, Quaternary; Protein Subunits; Rhodobacter sphaeroides; X-Ray Diffraction

The competition between the P(+)Q(A)(-) --> PQ(A) charge recombination (P, bacteriochlorophyll pair acting as primary photochemical electron donor) and the electron transfer to the secondary quinone acceptor Q(A)(-)Q(B) --> Q(A)Q(B)(-) (Q(A) and Q(B), primary and secondary electron accepting quinones) was investigated in chromatophores of Rb. capsulatus, varying the temperature down to -65 degrees C. The analysis of the flash-induced pattern for the formation of P(+)Q(A)Q(B)(-) shows that the diminished yield, when lowering the temperature, is not due to a homogeneous slowing of the rate constant k(AB) of the Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer but to a distribution of conformations that modulate the electron transfer rate over more than 3 orders of magnitude. This distribution appears "frozen", as no dynamic redistribution was observed over time ranges > 10 s (below -25 degrees C). The kinetic pattern was analyzed to estimate the shape of the distribution of k(AB), showing a bell-shaped band on the high rate side and a fraction of "blocked" reaction centers (RCs) with very slow k(AB). When the temperature is lowered, the high rate band moves to slower rate regions and the fraction of blocked RCs increases at the expense of the high rate band. The RCs that recombine from the P(+)Q(A)Q(B)(-) state appear temporarily converted to a state with rapid k(AB), indicating that the stabilized state described by Kleinfeld et al. (Biochemistry 1984, 23, 5780-5786) is still accessible at -60 degrees C.

Topics: Bacteriochlorophylls; Benzoquinones; Cold Temperature; Cytochromes c2; Electrons; Kinetics; Photosynthetic Reaction Center Complex Proteins; Protein Conformation; Rhodobacter capsulatus; Spectrometry, Fluorescence; Thermodynamics; Ubiquinone

The photosynthetic reaction center (RC) from purple bacteria converts light into chemical energy. Although the RC shows two nearly structurally symmetric branches, A and B, light-induced electron transfer in the native RC occurs almost exclusively along the A-branch to a primary quinone electron acceptor Q(A). Subsequent electron and proton transfer to a mobile quinone molecule Q(B) converts it to a quinol, Q(B)H(2). We report the construction and characterization of a series of mutants in Rhodobacter sphaeroides designed to reduce Q(B) via the B-branch. The quantum efficiency to Q(B) via the B-branch Phi(B) ranged from 0.4% in an RC containing the single mutation Ala-M260 --> Trp to 5% in a quintuple mutant which includes in addition three mutations to inhibit transfer along the A-branch (Gly-M203 --> Asp, Tyr-M210 --> Phe, Leu-M214 --> His) and one to promote transfer along the B-branch (Phe-L181 --> Tyr). Comparing the value of 0.4% for Phi(B) obtained in the AW(M260) mutant, which lacks Q(A), to the 100% quantum efficiency for Phi(A) along the A-branch in the native RC, we obtain a ratio for A-branch to B-branch electron transfer of 250:1. We determined the structure of the most effective (quintuple) mutant RC at 2.25 A (R-factor = 19.6%). The Q(A) site did not contain a quinone but was occupied by the side chain of Trp-M260 and a Cl(-). In this structure a nonfunctional quinone was found to occupy a new site near M258 and M268. The implications of this work to trap intermediate states are discussed.

Topics: Bacteriochlorophylls; Benzoquinones; Binding Sites; Crystallization; Crystallography, X-Ray; Electron Transport; Kinetics; Lasers; Models, Chemical; Mutagenesis, Site-Directed; Oxidation-Reduction; Pheophytins; Photolysis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Spectrophotometry

The dark reduction of photooxidized bacteriochlorophyll (P+) by photoreduced secondary quinone acceptor (QB-) in isolated reaction centers (RC) from the bacterium Rhodobacter sphaeroides wild type and mutant strain SA(L223) depending on the duration of light activation of RC was studied. The kinetics of the dark reduction of P+ decreased with increasing light duration, which is probably due to conformational changes occurring under prolonged light activation in RC from the wild type bacterium. In RC from bacteria of the mutant strain in which protonatable amino acid Ser L223 near QB is substituted by Ala, the dependence of reduction kinetics of P+ on duration of light was not observed. Such dependence, however, became observable after addition of cryoprotectors, namely glycerol and dimethylsulfoxide, to the RC samples from the mutant strain. It was concluded that substitution of Ser L223 with Ala disturbs the native mechanism of electrostatic stabilization of the electron in the RC quinone acceptor site. At the same time, an additional modification of RC hydrogen bonds by glycerol and dimethylsulfoxide probably includes various possibilities for more effective time delay of the electron on QB.

Topics: Bacteriochlorophylls; Benzoquinones; Binding Sites; Electrons; Kinetics; Light; Mutation; Oxidation-Reduction; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Time Factors

Symmetry-related branches of electron-transfer cofactors-initiating with a primary electron donor (P) and terminating in quinone acceptors (Q)-are common features of photosynthetic reaction centers (RC). Experimental observations show activity of only one of them-the A branch-in wild-type bacterial RCs. In a mutant RC, we now demonstrate that electron transfer can occur along the entire, normally inactive B-branch pathway to reduce the terminal acceptor Q(B) on the time scale of nanoseconds. The transmembrane charge-separated state P(+)Q(B)(-) is created in this manner in a Rhodobacter capsulatus RC containing the F(L181)Y-Y(M208)F-L(M212)H-W(M250)V mutations (YFHV). The W(M250)V mutation quantitatively blocks binding of Q(A), thereby eliminating Q(B) reduction via the normal A-branch pathway. Full occupancy of the Q(B) site by the native UQ(10) is ensured (without the necessity of reconstitution by exogenous quinone) by purification of RCs with the mild detergent, Deriphat 160-C. The lifetime of P(+)Q(B)(-) in the YFHV mutant RC is >6 s (at pH 8.0, 298 K). This charge-separated state is not formed upon addition of competitive inhibitors of Q(B) binding (terbutryn or stigmatellin). Furthermore, this lifetime is much longer than the value of approximately 1-1.5 s found when P(+)Q(B)(-) is produced in the wild-type RC by A-side activity alone. Collectively, these results demonstrate that P(+)Q(B)(-) is formed solely by activity of the B-branch carriers in the YFHV RC. In comparison, P(+)Q(B)(-) can form by either the A or B branches in the YFH RC, as indicated by the biexponential lifetimes of approximately 1 and approximately 6-10 s. These findings suggest that P(+)Q(B)(-) states formed via the two branches are distinct and that P(+)Q(B)(-) formed by the B side does not decay via the normal (indirect) pathway that utilizes the A-side cofactors when present. These differences may report on structural and energetic factors that further distinguish the functional asymmetry of the two cofactor branches.

Topics: Amino Acid Substitution; Bacteriochlorophylls; Benzoquinones; Electron Transport; Energy Metabolism; Imidoesters; Kinetics; Light-Harvesting Protein Complexes; Mutagenesis, Site-Directed; Oxidation-Reduction; Pheophytins; Photosynthetic Reaction Center Complex Proteins; Plasmids; Rhodobacter capsulatus; Spectrophotometry; Structure-Activity Relationship; Titrimetry; Triazines

We report time-resolved optical measurements of the primary electron transfer reactions in Rhodobacter capsulatus reaction centers (RCs) having four mutations: Phe(L181) --> Tyr, Tyr(M208) --> Phe, Leu(M212) --> His, and Trp(M250) --> Val (denoted YFHV). Following direct excitation of the bacteriochlorophyll dimer (P) to its lowest excited singlet state P, electron transfer to the B-side bacteriopheophytin (H(B)) gives P(+)H(B)(-) in approximately 30% yield. When the secondary quinone (Q(B)) site is fully occupied, P(+)H(B)(-) decays with a time constant estimated to be in the range of 1.5-3 ns. In the presence of excess terbutryn, a competitive inhibitor of Q(B) binding, the observed lifetime of P(+)H(B)(-) is noticeably longer and is estimated to be in the range of 4-8 ns. On the basis of these values, the rate constant for P(+)H(B)(-) --> P(+)Q(B)(-) electron transfer is calculated to be between approximately (2 ns)(-)(1) and approximately (12 ns)(-)(1), making it at least an order of magnitude smaller than the rate constant of approximately (200 ps)(-)(1) for electron transfer between the corresponding A-side cofactors (P(+)H(A)(-) --> P(+)Q(A)(-)). Structural and energetic factors associated with electron transfer to Q(B) compared to Q(A) are discussed. Comparison of the P(+)H(B)(-) lifetimes in the presence and absence of terbutryn indicates that the ultimate (i.e., quantum) yield of P(+)Q(B)(-) formation relative to P is 10-25% in the YFHV RC.

Topics: Bacteriochlorophylls; Benzoquinones; Dimerization; Electron Transport; Histidine; Imidoesters; Kinetics; Light-Harvesting Protein Complexes; Mutation; Nanotechnology; Phenylalanine; Pheophytins; Photolysis; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Static Electricity; Triazines; Tyrosine

The action of dipyridamole (DIP) on dark recombination between the photooxidized special pair bacteriochlorophyll BChl2+ and reduced primary quinone acceptor Q(A)- in the reaction centres (RCs) of the bacteria Rhodobacter sphaeroides was studied in the presence of different detergents (LDAO, Triton X-100, sodium cholate, sodium dodecyl sulfate). DIP accelerated this reaction approximately 4-5-fold. In RCs with the extracted H-subunit, the effect of DIP was observed at lower concentrations. The possibility of modification of the RC structure-dynamic state by DIP (including changes in RC hydrogen bonds) is proposed. The modification obviously disturbs the processes of the long-life electrostatic stabilization of Q(A)-.

Topics: Bacteriochlorophylls; Benzoquinones; Dipyridamole; Kinetics; Light-Harvesting Protein Complexes; Oxidation-Reduction; Phosphodiesterase Inhibitors; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides

Photosystem II (PSII) contains a collection of pheophytins (Pheo) and chlorophylls (Chl) that have unique absorbance spectra depending on their electronic structure and the surrounding protein environment. Despite numerous efforts to identify the spectra of each cofactor, differing assignments of the chromophore absorbance bands and electrochromic effects have led to conflicting models of pigment organization and chromophore interactions in PSII. We have utilized low-temperature measurements on well-defined redox states, together with the use of site-directed mutants, to make spectral assignments of several reaction center (RC) chromophores. Cryogenic (77 K) optical spectroscopy has been used to trap the bound redox-active quinone, Q(A), in the reduced form and measure the effect of the redox state of Q(A) on PSII chromophores without interference from other redox-active cofactors. The Q(A)(-) minus Q(A) difference spectrum contains a number of features that represent the perturbation of Pheo and Chl absorbance bands upon Q(A) reduction. Using site-directed mutants in which the axial ligand of the D1-side monomeric core Chl, P(A), is changed (D1-H198Q) or the hydrogen-bonding environment of the D1-side Pheo is modified (D1-Q130E), we have assigned the Q(y)() absorbance bands of four chromophores shifted by Q(A) reduction including both RC Pheos, the D1-side monomeric accessory Chl (B(A)), and one other Chl in PSII. The absorbance maximum of B(A) was identified at 683.5 nm from least-squares fits of the D1-H198Q minus wild type (WT) Q(A)(-) minus Q(A) double-difference spectrum; this assignment provides new evidence of a secondary effect of site-directed mutation on a RC chromophore. The other chromophores were assigned from simultaneous fits of the WT and D1-Q130E spectra in which the parameters of only the D1-side Pheo were allowed to vary. The D1-side and D2-side Pheos were found to have lambda(max) values at 685.6 and 669.3 nm, respectively, and another Chl influenced by Q(A)(-) was identified at 678.8 nm. These assignments are in good agreement with previous spectral analyses of intact PSII preparations and reveal that the number of chromophores affected by Q(A) reduction has been underestimated previously. In addition, the assignments are generally consistent with chromophore positions that are similar in the PSII RC and the bacterial photosynthetic RC.

Topics: Bacteriochlorophylls; Benzoquinones; Cold Temperature; Cyanobacteria; Electron Spin Resonance Spectroscopy; Free Radicals; Freezing; Glutamine; Histidine; Light-Harvesting Protein Complexes; Mutagenesis, Site-Directed; Oxidation-Reduction; Photosynthetic Reaction Center Complex Proteins; Photosystem II Protein Complex; Spectrophotometry; Tyrosine

The effect of Dipyridamole (10(-6)-10(-3) M) on the photomobilized electron transport in the system of quinone acceptors Q(A)-Q(B) of isolated photosynthetic reaction centers of Rhodobacter sphaeroides and on its temporary stabilization on Q(B) was studied. Depending on the type of the detergent present in the reaction center (lauryl dimethylamine oxide, Triton X-100, sodium dodecyl sulfate, and sodium cholate), dipyridamole could increase the time of the electron transfer to Q(B). The dipyridamole effect on the efficiency of the electron stabilization on Q(B) for reaction centers with different detergents was revealed in slowing down the process of dark reduction of photoactive bacteriochlorophyll from Q(B) at initial concentrations of added dipyridamole (10(-6)-10(-5) M) with following acceleration of the process at the dipyridamole concentrations of 10(-4)-10(-3) M. The pH lowering from 6.8-7.0 to 5.9-6.0 increased the dipyridamole effect. The possibility of the dipyridamole effect on the structural-dynamic state of the reaction center complex, including its hydrogen bond system, which influences the studied parameters of functional activity, is suggested.

Topics: Bacteriochlorophylls; Benzoquinones; Dipyridamole; Light-Harvesting Protein Complexes; Phosphodiesterase Inhibitors; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Structure-Activity Relationship

The effects of D2O, glycerol and dimethyl sulfoxide (DMSO) on redox potential Em of bacteriochlorophyll of a special P2 or [P(M)P(L)] pair, the rate of energy migration from bacteriopheophytin H(M) to [P(M)P(L)], electron transfer from [P(M)P(L)] to bacteriopheophytin H(L) and then to quinone Q(A) in reaction centers (RC) of Rhodobacter sphaeroides were studied. The H2O --> D2O substitution did not change Em of the special pair, whereas addition of 70% glycerol or 35% DMSO (v/v) increased the values of Em by 30 and 45 mV, respectively. Rate constants of energy migration km(H(M)* (km)--> P2), charge separation ke([P(M)P(L)] *H(L) (ke)--> [P(M)P(L)] +H(L)-), electron transfer to quinone kQ did not change after the glycerol addition, whereas isotopic substitution and addition of DMSO caused a 2-3-fold increase in km, ke, and kQ values. Theoretical analysis of the redox center potential dependence on dielectric permeability epsilon, swelling of the protein globule in a solvent, and on changes in the charge distribution (charge shifts) in the protein interior near the redox center was carried out. It has been shown that the H2O replacement with DMSO can result in the Em increase by tens of mV. No correlation was found between the Em values and the rate of charge separation upon isotopic substitution and addition of cryoprotectants. The effect of epsilon of the medium on the rate of electron transport due to changes of energy of intermolecular interaction between the donor and acceptor molecules was estimated.

Topics: Bacteriochlorophylls; Benzoquinones; Cryoprotective Agents; Deuterium; Electron Transport; Energy Metabolism; Kinetics; Light-Harvesting Protein Complexes; Models, Chemical; Models, Theoretical; Oxidation-Reduction; Photosynthetic Reaction Center Complex Proteins; Rhodobacter sphaeroides; Water

We report the primary charge separation events in a series of Rhodobacter capsulatus reaction centers (RCs) that have been genetically modified to contain a lysine near the bacteriochlorophyll molecule, BChl(M), on the nonphotoactive M-side of the RC. Using wild type and previously constructed mutants as templates, we substituted Lys for the native Ser residue at position 178 on the L polypeptide to make the S(L178)K single mutant, the S(L178)K/G(M201)D and S(L178)K/L(M212)H double mutants, and the S(L178)K/G(M201)D/L(M212)H triple mutant. In the triple mutant, the decay of the photoexcited primary electron donor (P) occurs with a time constant of 15 ps and is accompanied by 15% return to the ground state, 62% electron transfer to the L-side bacteriopheophytin, BPh(L), and 23% electron transfer to the M-side analogue, BPh(M). The data supporting electron transfer to the M-side include bleaching of the Q(X) band of BPh(M) at 528 nm and a spectrally and kinetically resolved anion band with a maximum at 640 nm assigned to BPh(M)(-). The decay of these features and concomitant approximately 20% decay of bleaching of the 850 nm band of P give a P(+)BPh(M)(-) lifetime on the order of 1-2 ns that reflects deactivation to give the ground state. These data and additional findings are compared to those from parallel experiments on the G(M201)D/L(M212)H double mutant, in which 15% electron transfer to BPh(M) has been reported previously and is reproduced here. We also compare the above results with the primary electron-transfer processes in S(L178)K, S(L178)K/G(M201)D, and S(L178)K /L(M212)H RCs and with those for the L(M212)H and G(M201)D single mutants and wild-type RCs. The comparison of extensive results that track the primary events in these eight RCs helps to elucidate key factors underlying the directionality and high yield of charge separation in the bacterial photosynthetic RC.

Topics: Amino Acid Substitution; Anions; Aspartic Acid; Bacteriochlorophylls; Benzoquinones; Electron Transport; Histidine; Kinetics; Light-Harvesting Protein Complexes; Lysine; Mutagenesis, Site-Directed; Pheophytins; Photochemistry; Photosynthetic Reaction Center Complex Proteins; Rhodobacter capsulatus; Spectrum Analysis

Quinones caused quenching of Chl a fluorescence in native and model systems. Menadione quenched twofold the fluorescence of Chl a and BChl a in pea chloroplasts, chromatophores of purple bacteria, and liposomes at concentrations of 50-80 microM. To obtain twofold quenching in Triton X-100 micelles and in ethanol, the addition of 1.3 mM and 11 mM menadione was required, respectively. A proportional decrease in the lifetime and yield of Chl a fluorescence in chloroplasts, observed as the menadione concentration increased, is indicative of the efficient excitation energy transfer from bulk Chl to menadione. The decrease in the lifetime and yield of fluorescence was close to proportional in liposomes, but not in detergent micelles. The insensitivity of the menadione quenching effect to DCMU in chloroplasts, and similarity of its action in chloroplasts and liposomes indicate that menadione in chloroplasts interacts with antenna Chl, i.e., nonphotochemical quenching of fluorescence occurs.

Topics: Bacterial Chromatophores; Bacteriochlorophylls; Benzoquinones; Chlorophyll; Chlorophyll A; Chloroplasts; Diuron; Fluorescence; Liposomes; Micelles; Pisum sativum; Quinones; Rhodobacter sphaeroides; Rhodospirillum rubrum; Spectrometry, Fluorescence; Ubiquinone; Vitamin K