Modulation of the Primary Electron Transfer Rate in Photosynthetic Reaction Centers by Reduction of a Secondary Acceptor

Photosynthetic application of picosecond spectroscopic techniques to bacterial reaction centers has led to a much greater understanding of the chemical nature of the initial steps of photosynthesis. Within 10 ps after excitation, a charge transfer complex is formed between the primary donor, a “special pair” of bacteriochlorophyll molecules, and a transient acceptor involving bacteriopheophytin. This complex subsequently decays in about 120 ps by donating the electron to a metastable acceptor, a tightly bound quinone.

Recent experiments with conventional optical and ESR techniques have shown that when reaction centers are illuminated by a series of single turnover flashes in the presence of excess electron donors and acceptors, a stable, anionic ubisemiquinone is formed on odd flashes and destroyed on even flashes, suggesting that the acceptor region contains a second quinone that acts as a two-electron gate between the reaction center and subsequent electron transport events involving the quinone pool.

Utilizing standard picosecond techniques, we have examined the decay of the charge transfer complex in reaction centers in the presence of the stable semiquinone, formed by flash illumination with a dye laser 10 s before excitation by a picosecond pulse. In this state the decay rate for the charge transfer complex is considerably slower than when no electron is present in the quinone acceptor region. This indicates fairly strong coupling between constituents of the reaction center-quinone acceptor complex and may provide a probe into the relative positions of the various components.

     

Geometry for the Primary Electron Donor and the Bacteriopheophytin Acceptor in Rhodopseudomonas viridis Photosynthetic Reaction Centers

The tetrapyrrole electron donors and acceptors (bacteriochlorophyll, BCh; bacteriopheophytin, BPh) within the bacterial photosynthetic reaction center (RC) are arranged with a specific geometry that permits rapid (picosecond time scale) electron tunneling to occur between them. Here we have measured the angle between the molecular planes of the bacteriochlorophyll dimer (primary donor), B₂, and the acceptor bacteriopheophytin, H, by analyzing the dichroism of the absorption change associated with H reduction, formed by photoselection with RCs of Rhodopseudomonas viridis. This angle between molecular planes is found to be 60° ± 2. This means that the ultrafast electron tunneling must occur between donors and acceptors that are fixed by the protein to have a noncoplanar alignment. Nearly perpendicular alignments have been determined for other electron tunneling complexes involving RCs. These geometries can be contrasted with models proposed for heme-heme electron transfer complexes, which have emphasized that mutually parallel orientations should permit the most kinetically facile transfers.     

Electron transfer in reaction centers of Rhodobacter sphaeroides and Rhodobacter capsulatus monitored by fluorescence of the bacteriochlorophyll dimer

Spectral and kinetic characteristics of fluorescence from isolated reaction centers of photosynthetic purple bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus were measured at room temperature under rectangular shape of excitation at 810 nm. The kinetics of fluorescence at 915 nm reflected redox changes due to light and dark reactions in the donor and acceptor quinone complex of the reaction center as identified by absorption changes at 865 nm (bacteriochlorophyll dimer) and 450 nm (quinones) measured simultaneously with the fluorescence. Based on redox titration and gradual bleaching of the dimer, the yield of fluorescence from reaction centers could be separated into a time-dependent (originating from the dimer) and a constant part (coming from contaminating pigment (detached bacteriochlorin)). The origin was also confirmed by the corresponding excitation spectra of the 915 nm fluorescence. The ratio of yields of constant fluorescence over variable fluorescence was much smaller in Rhodobacter sphaeroides (0.15±0.1) than in Rhodobacter capsulatus (1.2±0.3). It was shown that the changes in fluorescence yield reflected the disappearance of the dimer and the quenching by the oxidized primary quinone. The redox changes of the secondary quinone did not have any influence on the yield but excess quinone in the solution quenched the (constant part of) fluorescence. The relative yields of fluorescence in different redox states of the reaction center were tabulated. The fluorescence of the dimer can be used as an effective tool in studies of redox reactions in reaction centers, an alternative to the measurements of absorption kinetics.Abbreviations Bchl bacteriochlorophyll - Bpheo bacteriopheophytin - D electron donor to P⁺ - P bacteriochlorophyll dimer - Q quinone acceptor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ₆ ubiquinone-30     

Primary photochemistry of reaction centers from the photosynthetic purple bacteria

Photosynthetic organisms transform the energy of sunlight into chemical potential in a specialized membrane-bound pigment-protein complex called the reaction center. Following light activation, the reaction center produces a charge-separated state consisting of an oxidized electron donor molecule and a reduced electron acceptor molecule. This primary photochemical process, which occurs via a series of rapid electron transfer steps, is complete within a nanosecond of photon absorption. Recent structural data on reaction centers of photosynthetic bacteria, combined with results from a large variety of photochemical measurements have expanded our understanding of how efficient charge separation occurs in the reaction center, and have changed many of the outstanding questions.Abbreviations BChl bacteriochlorophyll - P a dimer of BChl molecules - BPh bacteriopheophytin - Q_A and Q_B quinone molecules - L, M and H light, medium and heavy polypeptides of the reaction center     

Studies of Electron Transport Dynamics in Photosynthetic Reaction Centers Using Fast Temperature Changes

Rates of thermoinduced conformational transitions of reaction center (RC) complexes providing effective electron transport were studied in chromatophores and isolated RC preparations of various photosynthesizing purple bacteria using methods of fast freezing and laser-induced temperature jump. Reactions of electron transfer from the primary to secondary quinone acceptors and from the multiheme cytochrome c subunit to photoactive bacteriochlorophyll dimer were used as probes of electron transport efficiency. The thermoinduced transition of the acceptor complex to the conformational state facilitating electron transfer to the secondary quinone acceptor was studied. It was shown that neither the characteristic time of the thermoinduced transition within the temperature range 233-253 K nor the characteristic time of spontaneous decay of this state at 253 K exceeded several tens of milliseconds. In contrast to the quinone complex, the thermoinduced transition of the macromolecular RC complex to the state providing effective electron transport from the multiheme cytochrome c to the photoactive bacteriochlorophyll dimer within the temperature range 220-280 K accounts for tens of seconds. This transition is thought to be mediated by large-scale conformational dynamics of the macromolecular RC complex.     

The photochemical reaction center of the bacteriochlorophyll b-containing organism Thiocapsa pfennigii

A photochemical reaction-center preparation has been made from a second bacteriochlorophyll b-containing organism, Thiocapsa pfennigii. The reaction-center unit is thought to be composed of one P-960, four bacteriochlorophyll, two bacteriopheophytin, one carotenoid molecules and polypeptides of M_r 40000, 37000, 34000, 27000 and 26000 probably plus quinones and metal atoms. The preparation also contains a low-potential cytochrome c-555 and a high-potential cytochrome c-557 bound to the reaction center in a 3–4:2–3:1 molar ratio with respect to P-960. The 40 kDa subunit is associated with the cytochromes, while the 37, 34 and 27 + 26 kDa subunits are proposed to be equivalent to the H, M and L polypeptides of bacteriochlorophyll a-containing reaction centers. The cytochromes are oxidized by P-960⁺. The three near-infrared absorption bands at 788, 840 and 968 nm are assigned to bacteriopheophytin, bacteriochlorophyll and the primary donor (P-960), respectively. The 778 nm peak resolves into two at 77 K; no further resolution of the other two peaks occurs. Illumination of the sodium dithionite-reduced reaction centers at 77 K by 960 nm-light results in P-960, transferring one electron from cytochrome c-555 mainly to a bacteriopheophytin molecule, absorbing at 781 nm. A similar treatment at room temperatures reduces most of the two bacteriopheophytin molecules. It is argued that both bacteriopheophytin molecules, possibly with some contribution from bacteriochlorophyll, form an intermediary electron-carrier complex between P-960 and a quinone in T. pfennigii. We could not substantiate that a bacteriochlorophyll molecule precedes the bacteriopheophytins in the electron transfer sequence. Although the biochemical characteristics of the reaction center are very similar to those of the other known bacterioclorophyll b-containing reaction center, that from Rhodopseudomonas viridis, their spectral characteristics are not. This has helped elucidate more about the function of each spectral form and led us to conclude that the 850 nm form in Rps. viridis is not the higher energy transition of the special pair of bacteriochlorophyll molecules forming P-960. Laser-flash-in-duced absorbance changes in T. pfennigii reaction-center preparation should now lead to a more complete understanding of the mechanism of the primary photochemical event.     

Investigation of the electron transfer reactions and redox characteristics of photoactive bacteriochlorophyll in Rhodobacter sphaeroides reaction centers modified by D2O and cryoprotectants

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.     

Spectroscopic properties of the intermediary electron carrier in the reaction center of Rhodopseudomonas viridis evidence for its interaction with the primary acceptor

The spectroscopic properties of the intermediary electron carrier (I), which functions between the bacteriochlorophyll dimer, (BChl)₂, and the primary acceptor quinone · iron, QFe, have been characterized in Rhodopseudomonas viridis. Optically the reduction of I is accompanied by a bleaching of bands at 545 and 790 nm and a broad absorbance increase around 680 nm which we attribute to the reduction of a bacteriopheophytin, together with apparent blue shifts of the bacteriochlorophyll bands at 830 and possibly at 960 nm. Low temperature electron paramagnetic resonance analysis also reveals complicated changes accompanying the reduction of I. In chromatophores I? is revealed as a broad split signal centered close to g 2.003, which is consistent with I? interacting, via exchange coupling and dipolar effects, with the primary acceptor Q?Fe. This is supported by experiments with reaction centers prepared with sodium dodecyl sulfate, which lack the Q?Fe g 1.82 signal, and also lack the broad split I? signal; instead, I? is revealed as an approximately 13 gauss wide free radical centered close to g 2.003. Reaction centers prepared using lauryl dimethylamine N-oxide retain most of their Q?Fe g 1.82 signal, and in this case I? occurs as a mixture of the two EPR signals described above. However, the optical changes accompanying the reduction of I? are very similar in the two reaction center preparations, so we conclude that there is no direct correlation between the two optical and the two EPR signals of I?. Perhaps the simplest explanation of the results is that the two EPR signals reflect the reduced bacteriopheophytin either interacting, or not interacting, with Q?Fe, while the optical changes reflect the reduction of bacteriophenophytin, together with secondary, perhaps electrochromic effects on the bacteriochlorophylls of the reaction center. However, we are unable to eliminate completely the possibility that there is also some electron sharing between the reduced bacteriopheophytin and bacteriochlorophyll.     

Why Is Electron Transport in the Reaction Centers of Purple Bacteria Unidirectional?

Although the two electron-transfer branches in the reaction centers (RC) of purple bacteria are virtually symmetric, it is well known that only one of them is functionally active (the A-branch). The mechanisms of functional asymmetry of structurally symmetric branches of the electron transport system are analyzed in this work within the framework of the theory of bimolecular charge-transfer complexes (CTC). CTC theory is shown to provide an explanation of this phenomenon. According to the CTC theory, the dominance of one branch is required to implement the CTC state in special bacteriochlorophyll pairs of RC, in which more than 30% of the excited electron density in the CTC is shifted toward one of the bacteriochlorophyll molecules. This causes a significant increase in the efficiency of further electron transfer to the primary quinone acceptor as compared to a system with two absolutely symmetric electron transfer branches. Specific features of dielectric asymmetry near the RC special pair are discussed. It is emphasized that a strong CTC is able to provide effective trapping of electronic excitation energy from antenna chlorophyll, which is a main function of the RC. Hypothetical stages of CTC formation in other classes of photosynthesizing bacteria during evolution are discussed.     

X-ray structure analysis of a membrane protein complex. Electron density map at 3 A resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis

X-ray analysis of three-dimensional crystals of the photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis led to an electron density distribution at 3 A resolution calculated with phases from multiple isomorphous replacement. The protein subunits of the complex were identified. An atomic model of the prosthetic groups of the reaction center complex (4 bacteriochlorophyll b, 2 bacteriopheophytin b. 1 non-heme iron, 1 menaquinone, 4 heme groups) was built. The arrangement of the ring systems of the bacteriochlorophyll b and bacteriopheophytin b molecules shows a local 2-fold rotation symmetry; two bacteriochlorophyll b form a closely associated, non-covalently linked dimer ("special pair"). A different local 2-fold symmetry axis is observed for the heme groups of the cytochrome part.     

Electron donors and acceptors in the initial steps of photosynthesis in purple bacteria: a personal account

The discovery by Louis N. M. Duysens in the 1950s that illumination of photosynthetic purple bacteria can cause oxidation of either a bacteriochlorophyll complex (P) or a cytochrome was followed by an extended period of uncertainty as to which of these processes was the `primary' photochemical reaction. Similar questions arose later about the roles of bacteriopheophytin (BPh) and quinones as the initial electron acceptor. This is a personal account of kinetic measurements that showed that electron transfer from P to BPh occurs in the initial step, and that the oxidized bacteriochlorophyll complex (P⁺) then oxidizes the cytochrome while the reduced BPh transfers an electron to a quinone. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dissipation in bioenergetic electron transfer chains

This paper examines the processes by which wasteful dissipation of free energy may occur in bioenergetic electron transfer chains. Frictionless transfer requires high rate constants in order to achieve a quasi-equilibrium steady-state. Previous results concerning the maximum power available from a photochemical source are recalled. The energetic performance of the bacterial reaction center is discussed, characterizing the processes that decrease either the quantum yield (recombination and obstruction) or the chemical potential (friction and non-equilibrated mechanisms). Considering the whole chain, diffusive carriers are potentially weaker links, due to kinetic limitation and short-circuiting reactions. It is suggested that the evolutionary trend has been to limit their number by lumping them into tightly bound protein complexes or, in a more flexible way, into labile supercomplexes.Abbreviations Cyt cytochrome - F Faraday - H primary acceptor in the bacterial reaction center (bacteriopheophytin) - k _B Boltzmann's constant - P primary photochemical donor (special bacteriochlorophyll pair) - RC reaction center - Q_A, Q_B primary, secondary quinone acceptor     

A picosecond-absorption study on bacteriochlorophyll excitation,trapping and primary-charge separation in chromatophores of Rhodospirillum rubrum

Absorbance-difference spectra and kinetics of absorbance changes were measured of chromatophores of Rhodospirillum rubrum by means of picosecond-absorption spectroscopy. A 35 ps excitation pulse at 532 nm produced absorbance changes due to the formation and decay of excited states of antenna pigments (Nuijs, A.M., Van Grondelle, R., Joppe, H.L.P., Van Bochove, A.C. and Duysens, L.N.M. (1985) Biochim. Biophys. Acta 810, 94–105), and, when open reaction centers were present, also those due to charge separation and primary electron transport. At low excitation energy density the lifetime of singlet-excited antenna bacteriochlorophyll was 80 ± 10 ps when the reaction centers were initially open and 200–400 ps when the primary electron donor was oxidized. Under the former conditions photooxidation of the primary donor occurred with a time constant of 70 ± 10 ps. Reduction of an electron-acceptor complex in the reaction center, probably involving both bacteriochlorophyll and bacteriopheophytin, was observed. Reoxidation of this acceptor occurred with a time constant of 200–300 ps. When the ubiquinone acceptor was reduced chemically, the primary radical pair decayed by recombination with a time constant of about 4 ns at high flash-energy densities, and of about 10 ns at lower energy densities. This dependence of the lifetime of the radical pair on the flash intensity was explained in terms of quenching processes by carotenoid triplet states in the antenna, and indicated a standard free-energy difference between the radical pair and the singlet-excited state of antenna bacteriochlorophyll of about 160 meV.     

Relation between structural-dynamic organization of reaction centers in Rhodobacter sphaeroides and picosecond steps of photosynthesis

The effect of deuteration, and the addition of glycerol and dimethylsulfoxide on the redox midpoint potential Em of bacteriochlorophyll of the special pair ?PMPL?, the rate of energy migration from bacteriopheophytin HM to ?PMPL?, and electron transfer from ?PMPL? to HL and from HL to quinone QA in reaction centers of Rhodobacter sphaeroides was studied. It was shown that H2O-->D2O substitution did not change Em of the special pair, while the addition of 70% glycerol and 35% dimethylsulfoxide (v/v) increased the Em value by 30 and 45 mV, correspondingly. The rate constants of energy migration [formula: see text], charge separation [formula: see text], electron transfer to QA kQ remained unchanged upon the addition of glycerol. The isotopic substitution of water and addition of dimethylsulfoxide led to a 2-3-fold increase in km, ke and kQ values. The dependence of the potential of redox center on the dielectric constant epsilon was analyzed. It was shown that replacement of H2O by dimethylsulfoxide can increase Em by tens of millivolt. There was no correlation between changes in Em and the values of km, ke and kQ upon deuteration and addition of cryoprotectors. It was concluded that the processes of energy migration, charge separation, and electron transfer to the quinone acceptor are preceded by the solvation of states H*M, ?P+MP-L?* and [formula: see text].     

Decrease of Fluorescence Intensity After the K Step in Chlorophyll a Fluorescence Induction is Suppressed by Electron Acceptors and Donors to Photosystem 2

Chlorophyll a fluorescence induction measured by a fluorometer with a high temperature stressed plant material shows a new K step which is a clear peak due to fast fluorescence rise and subsequent decrease of fluorescence intensity. We focused on an explanation of the decrease of fluorescence after the K step using artificial electron acceptors and donors to photosystem 2 (PS2). Addition of the artificial electron acceptors or donors suppressed the decrease of fluorescence after the K step. We suggest that the decrease mainly reflects (by more than 81 %) an energy loss process in the reaction centre of PS2 which is most probably a nonradiative charge recombination between P680⁺ (oxidised primary electron donor in PS2) and a negative charge stored on either Pheo⁻ or Q_A ⁻ (reduced primary electron acceptor of PS2 and reduced primary quinone electron acceptor of PS2, respectively). We suggest that the energy loss process is only possible when the inhibition of both the donor and the acceptor sides of PS2 occurs. This revised version was published online in August 2006 with corrections to the Cover Date.     

Primary photochemistry in the facultative green photosynthetic bacterium Chloroflexus aurantiacus

The mechanism of primary photochemistry has been investigated in purified cytoplasmic membranes and isolated reaction centers of Chloroflexus aurantiacus. Redox titrations on the cytoplasmic membranes indicate that the midpoint redox potential of P870, the primary electron donor bacteriochlorophyll, is +362 mV. An early electron acceptor, presumably menaquinone has Em 8.1 = -50 mV, and a tightly bound photooxidizable cytochrome c554 has Em 8.1 = +245 mV. The isolated reaction center has a bacteriochlorophyll to bacteriopheophytin ratio of 0.94:1. A two-quinone acceptor system is present, and is inhibited by o-phenanthroline. Picosecond transient absorption and kinetic measurements indicate the bacteriopheophytin and bacteriochlorophyll form an earlier electron acceptor complex.     

Relationship between the oxidation potential of the bacteriochlorophyll dimer and electron transfer in photosynthetic reaction centers

The primary electron donor in the photosynthetic reaction center from purple bacteria is a bacteriochlorophyll dimer containing four conjugated carbonyl groups that may form hydrogen bonds with amino acid residues. Spectroscopic analyses of a set of mutant reaction centers confirm that hydrogen bonds can be formed between each of these carbonyl groups and histidine residues in the reaction center subunits. The addition of each hydrogen bond is correlated with an increase in the oxidation potential of the dimer, resulting in a 355-mV range in the midpoint potential. The resulting changes in the free-energy differences for several reactions involving the dimer are related to the electron transfer rates using the Marcus theory. These reactions include electron transfer from cytochrome c₂ to the oxidized dimer, charge recombination from the primary electron acceptor quinone, and the initial forward electron transfer.     

The orientations of transition moments in reaction centers of Rhodopseudomonas sphaeroides, computed from data of linear dichroism and photoselection measurements.

Linear dichroism measurements of reaction centers of Rhodopseudomonas sphaeroides in stretched gelatin films have yielded angles that various optical transition moments make with an axis of symmetry in the reaction center. Photoselection experiments have yielded angles that certain transition moments make with each other. We have combined these data so as to compute the orientations of the Qx and Qy transition moments of the two molecules of bacteriopheophytin and of the bacteriochlorophyll special pair (photochemical electron donor) in the reaction center. Orientations are expressed in spherical polar coordinates with the symmetry axis as the pole. We have also computed additional angles between pairs of transition moments. In this treatment we have assumed that the bacteriopheophytins are independent monomers with little or no exciton coupling.     

Effect of reduction of the reaction center intermediate upon the picosecond oxidation reaction of the bacteriochlorophyll dimer in Chromatium vinosum and Rhodopseudomonas viridis

The photo-oxidation of the reaction center bacteriochlorophyll dimer or special pair was monitored at 1235 nm in Chromatium vinosum and at 1301 nm in Rhodopseudomonas viridis. In both species, the photo-oxidation was apparently complete within 10 ps after light excitation and proceeded unimpeded at low temperatures regardless of the prior state of reduction of the traditional primary electron acceptor, a quinone-iron complex. Thus the requirement for an intermediary electron carrier (I), previously established by picosecond measurements in Rps. sphaeroides (see ref. 4), is clearly a more general phenomenon.

The intermediary carrier, which involves bacteriopheophytin, was examined from the standpoint of its role as the direct electron acceptor from the photo-excited reaction center bacteriochlorophyll dimer. To accomplish this, the extent of light induced bacteriochlorophyll dimer oxidation was measured directly by the picosecond response of the infrared bands and indirectly by EPR assay of the triplet/biradical, as a function of the state of reduction of the I/I couple (measured by EPR) prior to activation. Two independent methods of obtaining I in a stably reduced form were used: chemical equilibrium reduction, and photochemical reduction. In both cases, the results demonstrated that the intermediary carrier, which we designate I, alone governs the capability for reaction center bacteriochlorophyll photooxidation, and as such I appears to be the immediate and sole electron acceptor from the light excited reaction center bacteriochlorophyll dimer.     

Structural basis of the drastically increased initial electron transfer rate in the reaction center from a Rhodopseudomonas viridis mutant described at 2.00-A resolution

It has previously been shown that replacement of the residue His L168 with Phe (HL168F) in the Rhodopseudomonas viridis reaction center (RC) leads to an unprecedented drastic acceleration of the initial electron transfer rate. Here we describe the determination of the x-ray crystal structure at 2.00-A resolution of the HL168F RC. The electron density maps confirm that a hydrogen bond from the protein to the special pair is removed by this mutation. Compared with the wild-type RC, the acceptor of this hydrogen bond, the ring I acetyl group of the "special pair" bacteriochlorophyll, D(L), is rotated, and its acetyl oxygen is found 1.1 A closer to the bacteriochlorophyll-Mg(2+) of the other special pair bacteriochlorophyll, D(M). The rotation of this acetyl group and the increased interaction between the D(L) ring I acetyl oxygen and the D(M)-Mg(2+) provide the structural basis for the previously observed 80-mV decrease in the D(+)/D redox potential and the drastically increased rate of initial electron transfer to the accessory bacteriochlorophyll, B(A). The high quality of the electron density maps also allowed a reliable discussion of the mode of binding of the triazine herbicide terbutryn at the binding site of the secondary quinone, Q(B).