Resonance Raman characterization of Rhodobacter sphaeroides reaction centers bearing site-directed mutations at tyrosine M210

Resonance Raman (RR) spectroscopy and low-temperature absorption spectroscopy have been used to investigate the structural changes in the reaction centers (RCs) of Rhodobacter sphaeroides induced by site-specific mutations on the tyrosine (Y) M210 residue. RCs in which Y M210 has been genetically replaced with phenylalanine (F) or leucine (L) exhibit a 5-fold decrease in their primary electron-transfer kinetics (Finkele et al., 1990). The general similarity of RR spectra of the wild-type RCs as compared to those of the two mutant RCs indicates that no significant global structural changes occur upon mutation at the level of any of the six bacteriochlorin pigments. In the RR spectra of the two mutant RCs there is a conspicuous absence of contributions from the BPheM prosthetic group, which is interpreted in terms of a change in the resonance enhancement conditions of this chromophore. Low-temperature adsorption spectroscopy reveals marked shifts in the Qx absorption band of BPheM. This shift is interpreted as arising from a destabilization of the protein in the vicinity of BPheM and accounts for the change in resonance condition for this chromophore in its RR contributions. As well, there is a 3-nm red shift of the Qy absorption band of the BChls from 803 to 806 nm for the mutant RCs. Difference RR spectra yielding structural information concerning, selectively, the primary donor (P) indicate that the structure of the P binding pocket is conserved for these mutant RCs. The tyrosine M210 is not observed to be engaged in a hydrogen bond with either of the acetyl or keto carbonyls of P.     

Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides

The kinetics of charge recombination following photoexcitation by a laser pulse have been analyzed in the reaction center-light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides. In RC-LH1 core complexes isolated from photosynthetically grown cells P(+)Q(B)(-) recombines with an average rate constant, k approximately 0.3 s(-1), more than three times smaller than that measured in RC deprived of the LH1 (k approximately 1 s(-1)). A comparable, slowed recombination kinetics is observed in RC-LH1 complexes purified from a pufX-deleted strain. Slowing of the charge recombination kinetics is even more pronounced in RC-LH1 complexes isolated from wild-type semiaerobically grown cells (k approximately 0.2 s(-1)). Since the kinetics of P(+)Q(A)(-) recombination is unaffected by the presence of the antenna, the P(+)Q(B)(-) state appears to be energetically stabilized in core complexes. Determinations of the ubiquinone-10 (UQ(10)) complement associated with the purified RC-LH1 complexes always yield UQ(10)/RC ratios larger than 10. These quinone molecules are functionally coupled to the RC-LH1 complex, as judged from the extent of exogenous cytochrome c(2) rapidly oxidized under continuous light excitation. Analysis of P(+)Q(B)(-) recombination, based on a kinetic model which considers fast quinone equilibrium at the Q(B) binding site, indicates that the slowing down of charge recombination kinetics observed in RC-LH1 complexes cannot be explained solely by a quinone concentration effect and suggests that stabilization of the light-induced charge separation is predominantly due to interaction of the Q(B) site with the LH1 complex. The high UQ(10) complements detected in RC-LH1 core complexes, but not in purified light-harvesting complex 2 and in RC, are proposed to reflect an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.     

Primary charge separation routes in the BChl:BPhe heterodimer reaction centers of Rhodobacter sphaeroides.

Energy transfer and the primary charge separation process are studied as a function of excitation wavelength in membrane-bound reaction centers of Rhodobacter sphaeroides in which the excitonically coupled bacteriochlorophyll homodimer is converted to a bacteriochlorophyll-bacteriopheophytin heterodimer, denoted D [Bylina, E. J., and Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S. A. 85, 7226]. In the HM202L heterodimer reaction center, excitation of D using 880 nm excitation light results in a 43 ps decay of the excited heterodimer, D. The decay of D results for about 30% in the formation of the charge separated state D+QA- and for about 70% in a decay directly to the ground state. Upon excitation of the monomeric bacteriochlorophylls using 798 nm excitation light, approximately 60% of the excitation energy is transferred downhill to D, forming D. Clear evidence is obtained that the other 40% of the excitations results in the formation of D+QA- via the pathway BA --> BA+HA- --> D+HA- --> D+QA-. In the membrane-bound "reversed" heterodimer reaction center HL173L, the simplest interpretation of the transient absorption spectra following B excitation is that charge separation occurs solely via the slow D-driven route. However, since a bleach at 812 nm is associated with the spectrum of D in the HL173L reaction center, it cannot be excluded that a state including BB is involved in the charge separation process in this complex.     

Femtosecond spectroscopy of primary charge separation in modified reaction centers of Rhodobacter sphaeroides (R-26)

Femtosecond measurements of kinetics and spectra of absorbance changes (ΔA) were carried out with modified reaction centers (RCs) from Rhodobacter sphaeroides (R-26) from which nonactive bacteriochlorophyll BM (located in the M protein subunit) was removed. The band of BM at 800 nm in native RCs is shifted in femtosecond measurements and obscures the ΔA of active bacteriochlorophyll BL (L subunit). The spectrum of ΔA in modified RCs at 6 ps delay includes the bleachings of the bands of P (primary electron donor) at 870 nm, of BL at 805 nm and of HL (bacteriopheophytin located in the L subunit) at 755 nm showing the reduction of 0̃.5 mol BL and 0̃.5 mol HL per mol P⁺. These data confirm an earlier suggestion that BL participates as an electron acceptor in the light-induced primary charge separation and agree with recent X-ray analysis of Rhodopseudomonas viridis and R. sphaeroides RCs which shows a location of BL between P and HL.     

Proton release upon oxidation of tyrosine in reaction centers from Rhodobacter sphaeroides

Markedly different light-induced protonational changes were measured in two reaction center mutants of Rhodobacter sphaeroides. A quadruple mutant containing alterations, at residues L131, M160, M197, and M210, that elevate the midpoint potential of the bacteriochlorophyll dimer was compared to the Y(M) mutant, which contains these alterations plus a tyrosine at M164 serving as a secondary electron donor [Kálmán et al., Nature 402 (1999) 696]. In the quadruple mutant, a proton uptake of 0.1-0.3 H(+)/reaction center between pH 6 and 10 resulted from formation of the oxidized bacteriochlorophyll donor and reduced primary quinone. In the Y(M) mutant, a maximal proton release of -0.5 H(+)/reaction center at pH 8 was attributed to formation of the tyrosyl radical and modeled using electrostatic and direct proton-releasing mechanisms.     

The effect of exchange of bacteriopheophytin a with plant pheophytin a on charge separation in Y(M210)W mutant reaction centers of Rhodobacter sphaeroides at low temperature

The bacteriopheophytin a molecules at the H(A) and H(B) binding sites of reaction centers (RCs) of the Y(M210)W mutant of Rhodobacter sphaeroides were chemically exchanged with plant pheophytin a. The Y(M210)W mutation slows down the formation of H(A)(-), presumably by raising the free energy level of the P(+)B(A)(-) state above that of P* due to increasing the oxidation potential of the primary electron donor P and lowering the reduction potential of the accessory bacteriochlorophyll B(A). Exchange of the bacteriopheophytins with pheophytin a on the contrary lowers the redox potential of H(A), inhibiting its reduction. A combination of the mutation and pigment exchange was therefore expected to make the A-side of the RC incapable of electron transfer and cause the excited state P* to deactivate directly to the ground state or through the B-side, or both. Time-resolved absorption difference spectroscopy at 10 K on the RCs that were modified in this way showed a lifetime of P* lengthened to about 500 ps as compared to about 200 ps measured in the original Y(M210)W RCs. We show that the decay of P* in the pheophytin-exchanged preparations is accompanied by both return to the ground state and formation of a new charge-separated state, the absorption difference spectrum of which is characterized by bleachings at 811 and 890 nm. This latter state was formed with a time constant of ca. 1.7 ns and a yield of about 30%, and lasted a few nanoseconds. On the basis of spectroscopic observations these bands at 811 and 890 nm are tentatively attributed to the presence of the P(+)B(B)(-) state, where B(B) is the accessory bacteriochlorophyll in the "inactive" B-branch of the cofactors. The B(B) molecules in Y(M210)W RCs are suggested to be spectrally heterogeneous, absorbing in the Q(y) region at 813 or 806 nm. The results are discussed in terms of perturbation of the free energy level of the P(+)B(B)(-) state and absorption properties of the B(B) bacteriochlorophyll in the mutant RCs due to a long-range effect of the Y(M210)W mutation on the protein environment of the B(B) binding pocket.     

Spectroscopic characterization of reaction centers of the (M)Y210W mutant of the photosynthetic bacterium Rhodobacter sphaeroides

The tyrosine-(M)210 of the reaction center of Rhodobacter sphaeroides 2.4.1 has been changed to a tryptophan using site-directed mutagenesis. The reaction center of this mutant has been characterized by low-temperature absorption and fluorescence spectroscopy, time-resolved sub-picosecond spectroscopy, and magnetic resonance spectroscopy. The charge separation process showed bi-exponential kinetics at room temperature, with a main time constant of 36 ps and an additional fast time constant of 5.1 ps. Temperature dependent fluorescence measurements predict that the lifetime of P^* becomes 4–5 times slower at cryogenic temperatures. From EPR and absorbance-detected magnetic resonance (ADMR, LD-ADMR) we conclude that the dimeric structure of P is not significantly changed upon mutation. In contrast, the interaction of the accessory bacteriochlorophyll B_A with its environment appears to be altered, possibly because of a change in its position.Abbreviations ADMR - absorbance-detected magnetic resonance - LDAO - N, N dimethyl dodecyl amine-N-oxide - RC - reaction center - LD-ADMR - linear-dichroic absorbance-detected magnetic resonance - P - primary donor - B - accessory bacteriochlorophyll - - bacteriopheophytin     

Qy-excitation resonance Raman scattering from the special pair in Rhodobacter sphaeroides reaction centers. Implications for primary charge separation.

Qy-excitation resonance Raman (RR) spectra are reported for reaction centers (RCs) from Rhodobacter sphaeroides 2.4.1. The RR spectra were acquired for both chemically reduced and oxidized RCs at 25 and 201 K by using a variety of excitation wavelengths in the range 800-920 nm. This range spans the Qy absorption bands of the special pair (P) and the accessory bacteriochlorophylls (BChls). The RR studies indicate that both P and the accessory BChls exhibit rich RR spectra in the 30-1800-cm-1 region. For both types of pigments, at least 20 bands are observed in the 30-750-cm-1 range. Although the frequencies of the modes of P and the accessory BChls are different, it is possible to make one-to-one correlations of the bands observed for the two types of pigments. This result suggests that the vibronically active low-frequency modes of P are derived from monomer-like vibrations (although they may be coupled monomer-like modes) rather than being vibrations resulting from the additional degrees of freedom present in the dimer. A plausible set of vibrational assignments for the low-frequency modes of both P and the accessory BChls is proposed on the basis of a semiempirical normal coordinate calculation. Comparison of the RR intensities of the low-frequency modes of P with those of the analogous modes of the accessory BChls indicates that the intensities of the modes of the former pigments are considerably larger than those of the latter. Collectively, the spectral data indicate that a large number of low-frequency modes of P are strongly coupled to the Qy electronic transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Manganese oxidation by modified reaction centers from Rhodobacter sphaeroides

The transfer of an electron from exogenous manganese (II) ions to the bacteriochlorophyll dimer, P, of bacterial reaction centers was characterized for a series of mutants that have P/P(+) midpoint potentials ranging from 585 to 765 mV compared to 505 mV for wild type. Light-induced changes in optical and EPR spectra of the mutants were measured to monitor the disappearance of the oxidized dimer upon electron donation by manganese in the presence of bicarbonate. The extent of electron transfer was strongly dependent upon the P/P(+) midpoint potential. The midpoint potential of the Mn(2+)/Mn(3+) couple was calculated to decrease linearly from 751 to 623 mV as the pH was raised from 8 to 10, indicating the involvement of a proton. The electron donation had a second order rate constant of approximately 9 x 10(4) M(-1) s(-1), determined from the linear increase in rate for Mn(2+) concentrations up to 200 microM. Weak dissociation constants of 100-200 microM were found. Quantitative EPR analysis of the six-line free Mn(2+) signal revealed that up to seven manganese ions were associated with the reaction centers at a 1 mM concentration of manganese. The association and the electron transfer between manganese and the reaction centers could be inhibited by Ca(2+) and Na(+) ions. The ability of reaction centers with high potentials to oxidize manganese suggests that manganese oxidation could have preceded water oxidation in the evolutionary development of photosystem II.     

Charge separation in Rhodobacter sphaeroides mutant reaction centers with increased midpoint potential of the primary electron donor

Primary charge separation dynamics in four mutant reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides with increased midpoint potential of the primary electron donor P (M160LH, L131LH, M197FH, and M160LH + L131LH + M197FH) have been studied by femtosecond transient absorption spectroscopy at room temperature. The decay of the excited singlet state in the wild-type and mutant RCs is complex and has two main exponential components, which indicates heterogeneity of electron transfer rates or the presence of reverse electron transfer reactions. The radical anion band of monomeric bacteriochlorophyll B_A at 1020 nm was first observed in transient absorbance difference spectra of single mutants. This band remains visible, although with somewhat reduced amplitude, even at delays up to tens of picoseconds when stimulated emission is absent and the reaction centers are in the P⁺H _A ^? state. The presence of this band in this time period indicates the existence of thermodynamic equilibrium between the P⁺B _A ^? H_A and P⁺B_AH _A ^? states. The data give grounds for assuming that the value of the energy difference between the states P*, P⁺B _A ^? H_A, and P⁺B_AH _A ^? at early times is of the same order of magnitude as the energy kT at room temperature. Besides, monomeric bacteriochlorophyll B_A is found to be an immediate electron acceptor in the single mutant RCs, where electron transfer is hampered due to increased energy of the P⁺B _A ^? state with respect to P*.     

Trapped tyrosyl radical populations in modified reaction centers from Rhodobacter sphaeroides

The photosynthetic reaction center from the purple bacterium Rhodobacter sphaeroides has been modified such that the bacteriochlorophyll dimer, when it becomes oxidized after light excitation, is capable of oxidizing tyrosine residues. One factor in this ability is a high oxidation-reduction midpoint potential for the dimer, although the location and protein environment of the tyrosine residue appear to be critical as well. These factors were tested in a series of mutants, each of which contains changes, at residues L131, M160, M197, and M210, that give rise to a bacteriochlorophyll dimer with a midpoint potential of at least 800 mV. The protein environment was altered near tyrosine residues that are either present in the wild type or introduced by mutagenesis, focusing on residues that could act as acceptors for the phenolic proton of the tyrosine upon oxidation. These mutations include Ser M190 to His, which is near Tyr L162, the combination of His M193 to Tyr and Arg M164 to His, which adds a Tyr-His pair, and the combinations of Arg L135 to Tyr with Tyr L164 to His, Arg L135 to Tyr with Tyr L144 to Glu, and Arg L135 to Tyr with Tyr L164 to Phe. Radicals were produced in the mutants by using light to initiate electron transfer. The radicals were trapped by freezing the samples, and the relative populations of the oxidized dimer and tyrosyl radicals were determined by analysis of low-temperature electron paramagnetic resonance spectra. The mutants all showed evidence of tyrosyl radical formation at high pH, and the extent of radical formation at Tyr L135 with pH differed depending on the identity of L144 and L164. The results show that tyrosine residues within approximately 10 A of the dimer can become oxidized when provided with a suitable protein environment.     

Small weak acids reactivate proton transfer in reaction centers from Rhodobacter sphaeroides mutated at AspL210 and AspM17

In reaction centers of Rhodobacter sphaeroides, site-directed mutagenesis has implicated several acidic residues in the delivery of protons to the secondary quinone (Q(B)) during reduction to quinol. In a double mutant (Asp(L210) --> Asn + Asp(M17) --> Asn) that is severely impaired in proton transfer capability over a wide pH range, proton transfer was "rescued" by added weak acids. For low pK(a) acids the total concentration of salt required near neutral pH was high. The ionic strength effect of added salts stimulated the rate of proton-coupled electron transfer at pH < 7, but decreased it at pH > 7.5, indicating an effective isoelectric point between these limits. In this region, a substantial rate enhancement by weak acids was clearly evident. A Br?nsted plot of activity versus pK(a) of the rescuing acids was linear, with a slope of -1, and extrapolated to a diffusion-limited rate at pK(a)(app) approximately 1. However, the maximum rate at saturating concentrations of acid did not correlate with pK(a), indicating that the acid and anion species compete for binding, both with weak affinity. This model predicts that pK(a)(app) corresponds to a true pK(a) = 4-5, similar to that for a carboxylic acid or Q(B)(-), itself. Only rather small, neutral acids were active, indicating a need to access a small internal volume, suggested to be a proton channel to the Q(B) domain. However, the on-rates were near the diffusion limit. The implications for intraprotein proton transfer pathway design are discussed.     

Effects of mutations near the bacteriochlorophylls in reaction centers from Rhodobacter sphaeroides.

Mutations were made in four residues near the bacteriochlorophyll cofactors of the photosynthetic reaction center from Rhodobacter sphaeroides. These mutations, L131 Leu to His and M160 Leu to His, near the dimer bacteriochlorophylls, and M203 Gly to Asp and L177 Ile to Asp, near the monomer bacteriochlorophylls, were designed to result in the placement of a hydrogen bond donor group near the ring V keto carbonyl of each bacteriochlorophyll. Perturbations of the electronic structures of the bacteriochlorophylls in the mutants are indicated by additional resolved transitions in the bacteriochlorophyll absorption bands in steady-state low-temperature and time-resolved room temperature spectra in three of the resulting mutant reaction centers. The major effect of the two mutations near the dimer was an increase up to 80 mV in the donor oxidation-reduction midpoint potential. Correspondingly, the calculated free energy difference between the excited state of the primary donor and the initial charge separated state decreased by up to 55 mV, the initial forward electron-transfer rate was up to 4 times slower, and the rate of charge recombination between the primary quinone and the donor was approximately 30% faster in these two mutants compared to the wild type. The two mutations near the monomer bacteriochlorophylls had minor changes of 25 mV or less in the donor oxidation-reduction potential, but the mutation close to the monomer bacteriochlorophyll on the active branch resulted in a roughly 3-fold decrease in the rate of the initial electron transfer.     

The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides

We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. M?ssbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS=1.06±0.01mm/s and QS=2.12±0.01mm/s), and Debye temperature (θ(D0)~167K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33meV, 33 and 42meV, and 53 and 60meV and a very sharp one at 44.5meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c(2), and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.     

Identification of the first steps in charge separation in bacterial photosynthetic reaction centers of Rhodobacter sphaeroides by ultrafast mid-infrared spectroscopy: electron transfer and protein dynamics

Time-resolved visible pump/mid-infrared (mid-IR) probe spectroscopy in the region between 1600 and 1800 cm⁻¹ was used to investigate electron transfer, radical pair relaxation, and protein relaxation at room temperature in the Rhodobacter sphaeroides reaction center (RC). Wild-type RCs both with and without the quinone electron acceptor Q_A, were excited at 600 nm (nonselective excitation), 800 nm (direct excitation of the monomeric bacteriochlorophyll (BChl) cofactors), and 860 nm (direct excitation of the dimer of primary donor (P) BChls (P_L/P_M)). The region between 1600 and 1800 cm⁻¹ encompasses absorption changes associated with carbonyl (CO) stretch vibrational modes of the cofactors and protein. After photoexcitation of the RC the primary electron donor P excited singlet state (P*) decayed on a timescale of 3.7 ps to the state (where B_L is the accessory BChl electron acceptor). This is the first report of the mid-IR absorption spectrum of ; the difference spectrum indicates that the 9-keto CO stretch of B_L is located around 1670-1680 cm⁻¹. After subsequent electron transfer to the bacteriopheophytin H_L in ∼1 ps, the state was formed. A sequential analysis and simultaneous target analysis of the data showed a relaxation of the radical pair on the ∼20 ps timescale, accompanied by a change in the relative ratio of the and bands and by a minor change in the band amplitude at 1640 cm⁻¹ that may be tentatively ascribed to the response of an amide CO to the radical pair formation. We conclude that the drop in free energy associated with the relaxation of , is due to an increased localization of the electron hole on the P_L half of the dimer and a further consequence is a reduction in the electrical field causing the Stark shift of one or more amide CO oscillators.     

Free energy dependence of the direct charge recombination from the primary and secondary quinones in reaction centers from Rhodobacter sphaeroides

The direct charge recombination rates from the primary quinone, k_AD (D⁺Q_A^– DQ_A) and the secondary quinone, k_BD (D⁺Q_B^– DQ_B), in reaction centers from Rhodobacter sphaeroides were measured as a function of the free energy differences for the processes, G_AD⁰ and G_BD⁰, respectively. Measurements were performed at 21 °C on a series of mutant reaction centers that have a wide range of dimer midpoint potentials and consequently a large variation in G_AD⁰ and G_BD⁰. As –G_AD⁰ varied from 0.43 to 0.78 eV, k_AD varied from 4.6 to 28.6 s^–1. The corresponding values for the wild type are 0.52 eV and 8.9 s^–1. Observation of the direct charge recombination rate k_BD was achieved by substitution of the primary quinone with naphthoquinones in samples in which ubiquinone was present at the secondary quinone site, resulting specifically in an increase in the free energy of the D⁺Q_A^– state relative to the D⁺Q_AQ_B^– state. As –G_BD⁰ varied from 0.37 to 0.67 eV, k_BD varied from 0.03 to 1.4 s^–1. The corresponding values for the wild type are 0.46 eV and 0.2 s^–1. A fit of the two sets of data to the Marcus theory for electron transfer yielded significantly different reorganization energies of 0.82 and 1.3 eV for k_AD and k_BD, respectively. In contrast, the fitted values for the coupling matrix element, or equivalently the maximum possible rate, were comparable (25 s^–1) for the two charge recombination processes. These results are in accord with Q_B having more interactions with dipoles, from both the surrounding protein and bound water molecules, than Q_A and with the primary determinant of the maximal rate being the quinone-donor distance.     

Strong effects of an individual water molecule on the rate of light-driven charge separation in the Rhodobacter sphaeroides reaction center

The role of a water molecule (water A) located between the primary electron donor (P) and first electron acceptor bacteriochlorophyll (B(A)) in the purple bacterial reaction center was investigated by mutation of glycine M203 to leucine (GM203L). The x-ray crystal structure of the GM203L reaction center shows that the new leucine residue packs in such a way that water A is sterically excluded from the complex, but the structure of the protein-cofactor system around the mutation site is largely undisturbed. The results of absorbance and resonance Raman spectroscopy were consistent with either the removal of a hydrogen bond interaction between water A and the keto carbonyl group of B(A) or a change in the local electrostatic environment of this carbonyl group. Similarities in the spectroscopic properties and x-ray crystal structures of reaction centers with leucine and aspartic acid mutations at the M203 position suggested that the effects of a glycine to aspartic acid substitution at the M203 position can also be explained by steric exclusion of water A. In the GM203L mutant, loss of water A was accompanied by an approximately 8-fold slowing of the rate of decay of the primary donor excited state, indicating that the presence of water A is important for optimization of the rate of primary electron transfer. Possible functions of this water molecule are discussed, including a switching role in which the redox potential of the B(A) acceptor is rapidly modulated in response to oxidation of the primary electron donor.     

X-ray crystal structure of the YM210W mutant reaction centre from Rhodobacter sphaeroides

The X-ray crystal structure of a reaction centre from Rhodobacter sphaeroides with a mutation of tyrosine M210 to tryptophan (YM210W) has been determined to a resolution of 2.5 A. Structural conservation is very good throughout the body of the protein, with the tryptophan side chain adopting a position in the mutant complex closely resembling that of the tyrosine in the wild-type complex. The spectroscopic properties of the YM210W reaction centre are discussed with reference to the structural data, with particular focus on evidence that the introduction of the bulkier tryptophan in place of the native tyrosine may cause a small tilt of the macrocycle of the B(L) monomeric bacteriochlorophyll.