Determination of proton transfer rates by chemical rescue: application to bacterial reaction centers

The bacterial reaction center (RC) converts light into chemical energy through the reduction of an internal quinone molecule Q(B) to Q(B)H(2). In the native RC, proton transfer is coupled to electron transfer and is not rate-controlling. Consequently, proton transfer is not directly observable, and its rate was unknown. In this work, we present a method for making proton transfer rate-controlling, which enabled us to determine its rate. The imidazole groups of the His-H126 and His-H128 proton donors, located at the entrance of the transfer pathways, were removed by site-directed mutagenesis (His --> Ala). This resulted in a reduction in the observed proton-coupled electron transfer rate [(Q(A)(-)(*)Q(B))Glu(-) + H(+) --> (Q(A)Q(B)(-)(*))GluH], which became rate-controlled by proton uptake to Glu-L212 [Adelroth, P., et al. (2001) Biochemistry 40, 14538-14546]. The proton uptake rate was enhanced (rescued) in a controlled fashion by the addition of imidazole or other amine-containing acids. From the dependence of the observed rate on acid concentration, an apparent second-order rate constant k((2)) for the "rescue" of the rate was determined. k((2)) is a function of the proton transfer rate and the binding of the acid. The dependence of k((2)) on the acid pK(a) (i.e., the proton driving force) was measured over 9 pK(a) units, resulting in a Br?nsted plot that was characteristic of general acid catalysis. The results were fitted to a model that includes the binding (facilitated by electrostatic attraction) of the cationic acid to the RC surface, proton transfer to an intermediate proton acceptor group, and subsequent proton transfer to Glu-L212. A proton transfer rate constant of approximately 10(5) s(-)(1) was determined for transfer from the bound imidazole group to Glu-L212 (over a distance of approximately 20 A). The same method was used to determine a proton transfer rate constant of 2 x 10(4) s(-)(1) for transfer to Q(B)(-)(*). The relatively fast proton transfer rates are explained by the presence of an intermediate acceptor group that breaks the process into sequential proton transfer steps over shorter distances. This study illustrates an approach that could be generally applied to obtain information about the individual rates and energies for proton transfer processes, as well as the pK(a)s of transfer components, in a variety of proton translocating systems.     

Identification of the proton pathway in bacterial reaction centers: decrease of proton transfer rate by mutation of surface histidines at H126 and H128 and chemical rescue by imidazole identifies the initial proton donors.

The pathway for proton transfer to Q(B) was studied in the reaction center (RC) from Rhodobacter sphaeroides. The binding of Zn(2+) or Cd(2+) to the RC surface at His-H126, His-H128, and Asp-H124 inhibits the rate of proton transfer to Q(B), suggesting that the His may be important for proton transfer [Paddock, M. L., Graige, M. S., Feher, G. and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188]. To assess directly the role of the histidines, mutant RCs were constructed in which either one or both His were replaced with Ala. In the single His mutant RCs, no significant effects were observed. In contrast, in the double mutant RC at pH 8.5, the observed rates of proton uptake associated with both the first and the second proton-coupled electron-transfer reactions k(AB)(()(1)()) [Q(A)(-)(*)Q(B)-Glu(-) + H(+) --> Q(A)(-)(*)Q(B)-GluH --> Q(A)Q(B)(-)(*)-GluH] and k(AB)(()(2)()) [Q(A)(-)(*)Q(B)(-)(*) + H(+) --> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)], were found to be slowed by factors of approximately 10 and approximately 4, respectively. Evidence that the observed changes in the double mutant RC are due to a reduction in the proton-transfer rate constants are provided by the observations: (i) k(AB)(1) at pH approximately pK(a) of GluH became biphasic, indicating that proton transfer is slower than electron transfer and (ii) k(AB)(2) became independent of the driving force for electron transfer, indicating that proton transfer is the rate-limiting step. These changes were overcome by the addition of exogenous imidazole which acts as a proton donor in place of the imidazole groups of His that were removed in the double mutant RC. Thus, we conclude that His-H126 and His-H128 facilitate proton transfer into the RC, acting as RC-bound proton donors at the entrance of the proton-transfer pathways.     

Observation of the protonated semiquinone intermediate in isolated reaction centers from Rhodobacter sphaeroides: implications for the mechanism of electron and proton transfer in proteins.

A proton-activated electron transfer (PAET) mechanism, involving a protonated semiquinone intermediate state, had been proposed for the electron-transfer reaction k(2)AB [Q(A)(-)(*)Q(B)(-)(*) + H(+) <--> Q(A)(-)(*)(Q(B)H)(*) --> Q(A)(Q(B)H)(-)] in reaction centers (RCs) from Rhodobacter sphaeroides [Graige, M. S., Paddock, M. L., Bruce, M. L., Feher, G., and Okamura, M. Y. (1996) J. Am. Chem. Soc. 118, 9005-9016]. Confirmation of this mechanism by observing the protonated semiquinone (Q(B)H)(*) had not been possible, presumably because of its low pK(a). By replacing the native Q(10) in the Q(B) site with rhodoquinone (RQ), which has a higher pK(a), we were able to observe the (Q(B)H)(*) state. The pH dependence of the semiquinone optical spectrum gave a pK(a) = 7.3 +/- 0.2. At pH < pK(a), the observed rate for the reaction was constant and attributed to the intrinsic electron-transfer rate from Q(A)(-)(*) to the protonated semiquinone (i.e., k(2)AB = k(ET)(RQ) = 2 x 10(4) s(-)(1)). The rate decreased at pH > pK(a) as predicted by the PAET mechanism in which fast reversible proton transfer precedes rate-limiting electron transfer. Consequently, near pH 7, the proton-transfer rate k(H) > 10(4) s(-)(1). Applying the two step mechanism to RCs containing native Q(10) and taking into account the change in redox potential, we find reasonable values for the fraction of (Q(B)H)(*) congruent with 0.1% (consistent with a pK(a)(Q(10)) of approximately 4.5) and k(ET)(Q(10)) congruent with 10(6) s(-)(1). These results confirm the PAET mechanism in RCs with RQ and give strong support that this mechanism is active in RCs with Q(10) as well.     

Identification of the proton pathway in bacterial reaction centers: cooperation between Asp-M17 and Asp-L210 facilitates proton transfer to the secondary quinone (QB)

The reaction center (RC) from Rhodobacter sphaeroides uses light energy to reduce and protonate a quinone molecule, Q(B) (the secondary quinone electron acceptor), to form quinol, Q(B)H2. Asp-L210 and Asp-M17 have been proposed to be components of the pathway for proton transfer [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. To test the importance of these residues for efficient proton transfer, the rates of the proton-coupled electron-transfer reaction k(AB)(2) (Q(A-*)Q(B-*) + H+ <==>Q(A-*)Q(B)H* --> Q(A)Q(B)H-) and its associated proton uptake were measured in native and mutant RCs, lacking one or both Asp residues. In the double mutant RCs, the k(AB)(2) reaction and its associated proton uptake were approximately 300-fold slower than in native RCs (pH 8). In contrast, single mutant RCs displayed reaction rates that were < or =3-fold slower than native (pH 8). In addition, the rate-limiting step of k(AB)(2) was changed from electron transfer (native and single mutants) to proton transfer (double mutant) as shown from the lack of a dependence of the observed rate on the driving force for electron transfer in the double mutant RCs compared to the native or single mutants. This implies that the rate of the proton-transfer step was reduced (> or =10(3)-fold) upon replacement of both Asp-L210 and Asp-M17 with Asn. Similar, but less drastic, differences were observed for k(AB)(1), which at pH > or =8 is coupled to the protonation of Glu-L212 [(Q(A-*)Q(B))-Glu- + H+ --> (Q(A)Q(B-*)-GluH]. These results show that the pathway for proton transfer from solution to reduced Q(B) involves both Asp-L210 and Asp-M17, which provide parallel branches to the proton-transfer pathway and through their electrostatic interaction have a cooperative effect on the proton-transfer rate. A possible mechanism for the cooperativity is discussed.     

Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases proton uptake by Glu-L212 upon first electron transfer to QB in reaction centers from Rhodobacter sphaeroides.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located approximately 5 A from the center of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 play an important role in the proton transfer to the Q(B) site [Paddock, M. L., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. (2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the amplitude of the proton uptake by Glu-L212 at pH 8 is increased in the single and double mutant RCs, as is evident from the larger intensity (by 35-55%) of the carboxylic acid band at 1727 cm(-1) in the Q(B)(-)/Q(B) difference spectra of mutant RCs, compared to that at 1728 cm(-1) in native RCs. This implies that the extent of ionization of Glu-L212 in the Q(B) ground state is greater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no changes in the protonation state or the environment of these two residues are detected upon Q(B) reduction. The absence of the 1727 cm(-1) signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 cm(-1) probes the protonation of Glu-L212 in native and mutant RCs.     

Retardation of proton transfer caused by binding of the transition metal ion to the bacterial reaction center is due to pKa shifts of key protonatable residues

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.     

Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy

Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.     

Revealing the involvement of extended hydrogen bond networks in the cooperative function between distant sites in bacterial reaction centers

In reaction center proteins of photosynthetic bacteria, the amplitude of proton uptake induced by the one-electron reduction of either of the two quinone electron acceptors (Q(A) and Q(B)) is an intrinsic observable of the electrostatic interactions associated with the redox function of the complex. We report here that, in Rhodobacter capsulatus, complete restoration of proton uptake (upon formation of Q(A)(-) and Q(B)(-)) to the level found in the wild type is observed in a mutant reaction center in which a tyrosine substitution in the Q(A) environment (Ala(M274) --> Tyr) is coupled with mutations of acidic residues near Q(B) (Glu(L212) --> Ala/Asp(L213) --> Ala) that initially cancel the proton uptake above pH 8. This result demonstrates that proton uptake occurs by strong cooperation between structural motifs, such as hydrogen-bonded networks, that span the 18 A distance between the two quinone acceptors.     

Proton and electron transfer in bacterial reaction centers

The bacterial reaction center couples light-induced electron transfer to proton pumping across the membrane by reactions of a quinone molecule Q(B) that binds two electrons and two protons at the active site. This article reviews recent experimental work on the mechanism of the proton-coupled electron transfer and the pathways for proton transfer to the Q(B) site. The mechanism of the first electron transfer, k((1))(AB), Q(-)(A)Q(B)-->Q(A)Q(-)(B), was shown to be rate limited by conformational gating. The mechanism of the second electron transfer, k((2))(AB), was shown to involve rapid reversible proton transfer to the semiquinone followed by rate-limiting electron transfer, H(+)+Q(-)(A)Q(-)(B) ifQ(-)(A)Q(B)H-->Q(A)(Q(B)H)(-). The pathways for transfer of the first and second protons were elucidated by high-resolution X-ray crystallography as well as kinetic studies showing changes in the rate of proton transfer due to site directed mutations and metal ion binding.     

Oxidation of the non-heme iron complex in photosystem II

In photosystem II (PSII), the redox properties of the non-heme iron complex (Fe complex) are sensitive to the redox state of quinones (Q(A/)(B)), which may relate to the electron/proton transfer. We calculated the redox potentials for one-electron oxidation of the Fe complex in PSII [E(m)(Fe)] based on the reference value E(m)(Fe) = +400 mV at pH 7 in the Q(A)(0)Q(B)(0) state, considering the protein environment in atomic detail and the associated changes in protonation pattern. Our model yields the pH dependence of E(m)(Fe) with -60 mV/pH as observed in experimental redox titration. We observed significant deprotonation at D1-Glu244 in the hydrophilic loop region upon Fe complex oxidation. The calculated pK(a) value for D1-Glu244 depends on the Fe complex redox state, yielding a pK(a) of 7.5 and 5.5 for Fe(2+) and Fe(3+), respectively. To account for the pH dependence of E(m)(Fe), a model involving not only D1-Glu244 but also the other titratable residues (five Glu in the D-de loops and six basic residues near the Fe complex) seems to be needed, implying the existence of a network of residues serving as an internal proton reservoir. Reduction of Q(A/B) yields +302 mV and +268 mV for E(m)(Fe) in the Q(A)(-)Q(B)(0) and Q(A)(0)Q(B)(-) states, respectively. Upon formation of the Q(A)(0)Q(B)(-) state, D1-His252 becomes protonated. Forming Fe(3+)Q(B)H(2) by a proton-coupled electron transfer process from the initial state Fe(2+)Q(B)(-) results in deprotonation of D1-His252. The two EPR signals observed at g = 1.82 and g = 1.9 in the Fe(2+)Q(A)(-) state of PSII may be attributed to D1-His252 with variable and fixed protonation, respectively.     

Identification of a novel protonation pattern for carboxylic acids upon Q(B) photoreduction in Rhodobacter sphaeroides reaction center mutants at Asp-L213 and Glu-L212 sites

In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule Q(B). Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon formation, Q(B)(-) induces protonation of Glu-L212, located within 5 A of Q(B), resulting in a IR signal at 1728 cm(-1). However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770-1700 cm(-1)). In particular, no signal for Asp-L213 is found despite its juxtaposition to Q(B) and importance for proton uptake on the second electron-transfer step. In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of Q(B) was modified by interchanging Asp and Glu at the L213 and L212 positions. The Q(B)(-)/Q(B) FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770-1700 cm(-1) range shows several distinct new signals, which are sensitive to (1)H/(2)H isotopic exchange, indicating that the reduction of Q(B) results in the change of the protonation state of several carboxylic acids. The new bands at 1752 and 1747 cm(-1) were assigned to an increase of protonation in response to Q(B) reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues. Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair. Implications for the native reaction center are discussed.     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.     

Anomalous acceleration of the photocycle in photosynthetic reaction centers inhibited on the acceptor side

The rate of the photocycle (quinone reduction cycle) was measured under continuous light excitation in an isolated reaction center protein of the photosynthetic bacterium Rhodobacter sphaeroides. The rate is determined by the slowest step of the photocycle, which could be the photochemistry (charge separation), the quinone/quinol and cytochrome c(2+)/c(3+) exchanges, or proton delivery to the secondary quinone. The photocycle was driven by high light intensity of a laser diode (5 W/cm(2) at 808 nm) to avoid light limitation of the observed rate. The fast turnover of the reaction center (up to 10(3) s(-1)) was slowed down by inhibition of the proton delivery to the secondary quinone by transition metal ions (Cd(2+) and Ni(2+)), by mutation of a key protonatable group (L213Asp --> Asn), or by use of low-affinity ubiquinone (UQ(0)) to the secondary quinone binding site. Although in all of these cases the rate of turnover was 2-3 orders of magnitude less than that of the primary photochemistry, marked light intensity dependence was observed. The rate of the photocycle increased from 7 s(-1) (Ni(2+), low light intensity) to 27 s(-1) (high light intensity) at pH 8.4. The anomalous reacceleration is due to alternative events on the acceptor side induced by continuous excitation. We argue that the continuous excitation of the protein trapped in the reduced acceptor (Q(A)(-)Q(B)(-)) state produces short-lived reduced bacteriopheophytin (I(-)) that delivers activation energy to anomalous changes on the acceptor side as second interquinone electron transfer before proton uptake or increase of the quinone dissociation constant.     

Rapid-scan Fourier transform infrared spectroscopy shows coupling of GLu-L212 protonation and electron transfer to Q(B) in Rhodobacter sphaeroides reaction centers

Rapid-scan Fourier transform infrared (FTIR) difference spectroscopy was used to investigate the electron transfer reaction Q(A-)Q(B)-->Q(A)Q(B-) (k(AB)(1)) in mutant reaction centers of Rhodobacter sphaeroides, where Asp-L210 and/or Asp-M17 have been replaced with Asn. Mutation of both residues decreases drastically k(AB)(1)), attributed to slow proton transfer to Glu-L212, which becomes rate limiting for electron transfer to Q(B) [M.L. Paddock et al., Biochemistry 40 (2001) 6893]. In the double mutant, the FTIR difference spectrum recorded during the time window 4-29 ms following a flash showed peaks at 1670 (-), 1601 (-) and 1467 (+) cm(-1), characteristic of Q(A) reduction. The time evolution of the spectra shows reoxidation of Q(A-) and concomitant reduction of Q(B) with a kinetics of about 40 ms. In native reaction centers and in both single mutants, formation of Q(B-) occurs much faster than in the double mutant. Within the time resolution of the technique, protonation of Glu-L212, as characterized by an absorption increase at 1728 cm(-1) [E. Nabedryk et al., Biochemistry 34 (1995) 14722], was found to proceed with the same kinetics as reduction of Q(B) in all samples. These rapid-scan FTIR results support the model of proton uptake being rate limiting for the first electron transfer from Q(A-) to Q(B) and the identification of Glu-L212 as the main proton acceptor in the state Q(A)Q(B-).     

Two groups control light-induced schiff base deprotonation and the proton affinity of asp(85) in the Arg(82)His mutant of bacteriorhodopsin

Arg(82) is one of the four buried charged residues in the retinal binding pocket of bacteriorhodopsin (bR). Previous studies show that Arg(82) controls the pK(a)s of Asp(85) and the proton release group and is essential for fast light-induced proton release. To further investigate the role of Arg(82) in light-induced proton pumping, we replaced Arg(82) with histidine and studied the resulting pigment and its photochemical properties. The main pK(a) of the purple-to-blue transition (pK(a) of Asp(85)) is unusually low in R82H: 1.0 versus 2.6 in wild type (WT). At pH 3, the pigment is purple and shows light and dark adaptation, but almost no light-induced Schiff base deprotonation (formation of the M intermediate) is observed. As the pH is increased from 3 to 7 the M yield increases with pK(a) 4.5 to a value approximately 40% of that in the WT. A transition with a similar pK(a) is observed in the pH dependence of the rate constant of dark adaptation, k(da). These data can be explained, assuming that some group deprotonates with pK(a) 4.5, causing an increase in the pK(a) of Asp(85) and thus affecting k(da) and the yield of M. As the pH is increased from 7 to 10.5 there is a further 2.5-fold increase in the yield of M and a decrease in its rise time from 200 &mgr;s to 75 &mgr;s with pK(a) 9. 4. The chromophore absorption band undergoes a 4-nm red shift with a similar pK(a). We assume that at high pH, the proton release group deprotonates in the unphotolyzed pigment, causing a transformation of the pigment into a red-shifted "alkaline" form which has a faster rate of light-induced Schiff base deprotonation. The pH dependence of proton release shows that coupling between Asp(85) and the proton release group is weakened in R82H. The pK(a) of the proton release group in M is 7.2 (versus 5.8 in the WT). At pH < 7, most of the proton release occurs during O --> bR transition with tau approximately 45 ms. This transition is slowed in R82H, indicating that Arg(82) is important for the proton transfer from Asp(85) to the proton release group. A model describing the interaction of Asp(85) with two ionizable residues is proposed to describe the pH dependence of light-induced Schiff base deprotonation and proton release.     

Exploring the energy profile of the Q(A)(-) to Q(B) electron transfer reaction in bacterial photosynthetic reaction centers: pH dependence of the conformational gating step

Both large- and small-scale conformational changes are needed as proteins carry out reactions. However, little is known about the identity, energy of, and barriers between functional substates on protein reaction coordinates. In isolated bacterial photosynthetic reaction centers, the electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B), is rate limited by conformational changes at low pH and by proton binding at high pH. The kinetics and thermodynamics of this reaction were determined between 200 and 300 K from pH 6 to pH 10.5. A model with two substates of the reactant, P(+)Q(A)(-)Q(B), one protonated (state A) and one unprotonated (alpha), and one state of the product, P(+)Q(A)Q(B)(-) (B), was able to simulate the dependence of the rate on temperature and pH fairly well. The equilibrium between the three states were measured in situ at each temperature. Proton binding (alpha to A transition) has a favorable DeltaH and unfavorable DeltaS as does the conformational changes required for electron transfer at low pH (A to B). The pK for the A to alpha transition is 9.7 at room temperature, consistent with previous measurements, and equivalent to 13.5 at 200 K. The activation barriers were determined for each transition. Both the alpha to A and the A to B transitions are limited primarily by the activation enthalpy with modest DeltaS.     

Evidence for the rate of the final step in the bacteriorhodopsin photocycle being controlled by the proton release group: R134H mutant

Light absorbed by bacteriorhodopsin (bR) leads to a proton being released at the extracellular surface of the purple membrane. Structural studies as well as studies of mutants of bR indicate that several groups form a pathway for proton transfer from the Schiff base to the extracellular surface. These groups include D85, R82, E204, E194, and water molecules. Other residues may be important in tuning the initial state pK(a) values of these groups and in mediating light-induced changes of the pK(a) values. A potentially important residue is R134: it is located close to E194 and might interact electrostatically to affect the pK(a) of E194 and light-induced proton release. In this study we investigated effects of the substitution of R134 with a histidine on light-induced proton release and on the photocycle transitions associated with proton transfer. By measuring the light-induced absorption changes versus pH, we found that the R134H mutation results in an increase in the pK(a) of the proton release group in both the M (0.6 pK unit) and O (0.7 pK unit) intermediate states. This indicates the importance of R134 in tuning the pK(a) of the group that, at neutral and high pH, releases the proton upon M formation (fast proton release) and that, at low pH, releases the proton simultaneously with O decay (slow proton release). The higher pK(a) of the proton release group found in R134H correlates with the slowing of the rate of the O --> bR transition at low pH and probably is the cause of this slowing. The pH dependence of the fraction of the O intermediate is altered in R134H compared to the WT but is similar to that in the E194D mutant: a very small amount of O is present at neutral pH, but the fraction of O increases greatly upon decreasing the pH. These results provide further support for the hypothesis that the O --> bR transition is controlled by the rate of deprotonation of the proton release group. These data also provide further evidence for the importance of the R134-E194 interaction in modulating proton release from D85 after light has led to its being protonated.     

Molecular anatomy of the alkaliphilic xylanase from Bacillus halodurans C-125

Two regions in xylanase A from Bacillus halodurans C-125 (XynA), an alkaliphilic xylanase, were identified to be responsible for its activity at basic pH by comparing the dissociation constants of the XynA proton donor Glu residue (pK(e2) and pK(es2)) with those of xylanase B from Clostridium stercorarium F9 (XynB) and their mutants constructed by substituting either Ser137/Asn127 of XynA/XynB or the 4th loop, designed based on the structural difference close to the proton donor. The substitution of XynB at Asn127 into Ser increased pK(e2) by 0.37. The effect is explained that the positive charge of His126 likely affects the proton donor via Asn127 and a water molecule in XynB, resulting in a decrease in pK(e2), whereas such interactions were not observed with Ser. The substitution of XynB at the 4th loop into XynA (XynB Loop4A) increased the pK(e2) and pK(es2) values by 0.29 and 0.62, respectively. The effect of the 4th loop in XynA is likely due to a hydrogen bond between Asp199 in the loop and Tyr239, which interacts with both the proton donors Glu195 and Arg204, with flexibility of the loop. Both the mutations independently affected the increases in pK(e2).     

Fourier transform infrared evidence for early deprotonation of Asp(85) at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm(-1), indicating that Asp(85) and Asp(96) are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm(-1) and a negative peak at 1742 cm(-1), revealing the presence of protonated Asp(85) (in an M intermediate environment) and deprotonated Asp(96). These results indicate that in E194Q-containing mutants, the second increase in the Asp(85) pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu(194) is the group that controls the pK(a) of Asp(85).