Energetics of Proton Transfer Pathways in Reaction Centers from Rhodobacter sphaeroides
2005; Elsevier BV; Volume: 280; Issue: 13 Linguagem: Inglês
10.1074/jbc.m413531200
ISSN1083-351X
AutoresHiroshi Ishikita, Ernst‐Walter Knapp,
Tópico(s)ATP Synthase and ATPases Research
ResumoElectron transfer between the primary and secondary quinones (QA, QB) in the bacterial photosynthetic reaction center (bRC) is coupled with proton uptake at QB. The protons are conducted from the cytoplasmic side, probably with the participation of two water channels. Mutations of titratable residues like Asp-L213 to Asn (inhibited mutant) or the double mutant Glu-L212 to Ala/Asp-L213 to Ala inhibit these electron transfer-coupled proton uptake events. The inhibition of the proton transfer (PT) process in the single mutant can be restored by a second mutation of Arg-M233 to Cys or Arg-H177 to His (revertant mutant). These revertant mutants shed light on the location of the main proton transfer pathway of wild type bRC. In contrast to the wild type and inhibited mutant bRC, the revertant mutant bRC showed notable proton uptake at Glu-H173 upon formation of the QB– state. In all of these mutants, the pKa of Asp-M17 decreased by 1.4–2.4 units with respect to the wild type bRC, whereas a significant pKa upshift of up to 5.8 units was observed at Glu-H122, Asp-H170, Glu-H173, and Glu-H230 in the revertant mutants. These residues belonging to the main PT pathway are arranged along water channel P1 localized mainly in subunit H. bRC possesses subunit H, which has no counterpart in photosystem II. Thus, bRC may possess alternative PT pathways involving water channels in subunit H, which becomes active in case the main PT pathway is blocked. Electron transfer between the primary and secondary quinones (QA, QB) in the bacterial photosynthetic reaction center (bRC) is coupled with proton uptake at QB. The protons are conducted from the cytoplasmic side, probably with the participation of two water channels. Mutations of titratable residues like Asp-L213 to Asn (inhibited mutant) or the double mutant Glu-L212 to Ala/Asp-L213 to Ala inhibit these electron transfer-coupled proton uptake events. The inhibition of the proton transfer (PT) process in the single mutant can be restored by a second mutation of Arg-M233 to Cys or Arg-H177 to His (revertant mutant). These revertant mutants shed light on the location of the main proton transfer pathway of wild type bRC. In contrast to the wild type and inhibited mutant bRC, the revertant mutant bRC showed notable proton uptake at Glu-H173 upon formation of the QB– state. In all of these mutants, the pKa of Asp-M17 decreased by 1.4–2.4 units with respect to the wild type bRC, whereas a significant pKa upshift of up to 5.8 units was observed at Glu-H122, Asp-H170, Glu-H173, and Glu-H230 in the revertant mutants. These residues belonging to the main PT pathway are arranged along water channel P1 localized mainly in subunit H. bRC possesses subunit H, which has no counterpart in photosystem II. Thus, bRC may possess alternative PT pathways involving water channels in subunit H, which becomes active in case the main PT pathway is blocked. The primary event in the bacterial photosynthetic reaction center (bRC) 1The abbreviations used are: bRC, bacterial photosynthetic reaction center; AA mutant, E(L212)A/D(L213)A mutant; ET, electron transfer; inhibited mutant, proton transfer inactive D(L213)N single mutant; PSII, photosystem II; PT, proton transfer; QA and QB, ubiquinone in the A- and B-branch, respectively; Q–, ubiquinone in the reduced state; Q0, ubiquinone in the neutral charge state; revertant mutant, proton transfer active D(L213)A/R(M233)C double mutant. after electronic excitation of the bacteriochlorophyll a dimer, the special pair, is a charge-separation process. As a result, the special pair becomes oxidized while an electron is transferred along the A-branch cofactors from an accessory bacteriochlorophyll a via bacteriopheophytin to ubiquinone QA in the A-branch and subsequently to QB in the B-branch. After the first ET process, Q –B is protonated and forms QBH that is stabilized by a second ET and proton transfer (PT) event, resulting in the formation of the doubly protonated dihydroquinone QBH2. Seven residues (namely His-H126, His-H128, Asp-M17, Asp-L210, Glu-L212, Asp-L213, and Ser-L223) were suggested to be involved in these PT events (reviewed in Ref. 1.Nabedryk E. Breton J. Okamura M.Y. Paddock M.L. Biochemistry. 2001; 40: 13826-13832Crossref PubMed Scopus (24) Google Scholar) (Fig. 1). The single mutant Asp-L213 to Asn (D(L213)N) decreases the rate of these PT events by about a factor of 106 (2.Paddock M.L. Senft M.E. Graige M.S. Rongey S.H. Turanchik T. Feher G. Okamura M.Y. Photosynth. Res. 1998; 55: 281-291Crossref Google Scholar). The decreased PT rate for the single mutant can be recovered by an additional mutation of Asn-M44 to Asp, since the side chain of Asp at M44 can substitute the removed carboxylate at L213 in the mutant bRC (3.Hanson D.K. Nance S.L. Schiffer M. Photosynth. Res. 1992; 32: 147-153Crossref PubMed Scopus (32) Google Scholar, 4.Rongey S.H. Paddock M.L. Feher G. Okamura M.Y. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1325-1329Crossref PubMed Scopus (62) Google Scholar, 5.Paddock M.L. Sagle L. Tehrani A. Beatty J.T. Feher G. Okamura M.Y. Biochemistry. 2003; 42: 9626-9632Crossref PubMed Scopus (28) Google Scholar). On the other hand, the PT in the D(L213)N mutant bRC was also restored by mutations of Arg-M233 to Cys or Arg-H177 to His, namely the double mutant D(L213)A/R(M233)C or D(L213)A/R(H177)H, respectively (2.Paddock M.L. Senft M.E. Graige M.S. Rongey S.H. Turanchik T. Feher G. Okamura M.Y. Photosynth. Res. 1998; 55: 281-291Crossref Google Scholar, 6.Hanson D.K. Baciou L. Tiede D.M. Nance S.L. Schiffer M. Sebban P. Biochim. Biophys. Acta. 1992; 1102: 260-265Crossref PubMed Scopus (60) Google Scholar, 7.Paddock M.L. Feher G. Okamura M.Y. FEBS Lett. 2003; 555: 45-50Crossref PubMed Scopus (104) Google Scholar). Similar revertants were also observed in the AA mutant bRC from Rhodobacter capsulatus (8.Maróti P. Hanson D.K. Baciou L. Schiffer M. Sebban P. Proc. Natl. Acad. Sci. U. S. A. 1995; 91: 5617-5621Crossref Scopus (59) Google Scholar). However, in these two mutants, no carboxylate is reintroduced, and, based on the wild type bRC structure, the corresponding two mutated sites M233 and H177 have a distance of more than 10 Å from residue L213 (i.e. 13 and 17 Å, respectively) (9.Stowell M.H.B. McPhillips T.M. Rees D.C. Solitis S.M. Abresch E. Feher E. Science. 1997; 276: 812-816Crossref PubMed Scopus (730) Google Scholar) (Fig. 1). Based on the observed structural changes close to Glu-H173 from wild type to revertant mutant bRC, the proposed mechanism to recover PT in the revertants involves Glu-H173 in the PT pathway (7.Paddock M.L. Feher G. Okamura M.Y. FEBS Lett. 2003; 555: 45-50Crossref PubMed Scopus (104) Google Scholar, 10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). In experimental investigations on ET-coupled proton uptake at QB, it is suggested that the addition of Cd2+ and Zn2+ ions decreases the rates of ET from QA to QB (11.Utschig L.M. Ohigashi Y. Thurnauer M.C. Tiede D.M. Biochemistry. 1998; 37: 8278-8281Crossref PubMed Scopus (66) Google Scholar, 12.Paddock M.L. Graige M.S. Feher G. Okamura M.Y. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6183-6188Crossref PubMed Scopus (116) Google Scholar). The crystal structure confirms that these metal ions bind at Asp-H124, His-H126, and His-H128 (13.Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 1542-1547Crossref PubMed Scopus (120) Google Scholar). Thus, these two histidines were proposed as the proton entry point. However, the rates of the first and second ET process in the H(H126)A/H(H128)A double mutant were reduced by only a factor of 10 and 4 relative to the wild type bRC (14.Adelroth P. Paddock M.L. Tehrani A. Beatty J.T. Feher G. Okamura M.Y. Biochemistry. 2001; 40: 14538-14546Crossref PubMed Scopus (66) Google Scholar). Alternatively, the metal binding effect on the ET rates was suggested to be due to electrostatic interactions rather than blocking of the proton entry point (15.Gerencser L. Maroti P. Biochemistry. 2001; 40: 1850-1860Crossref PubMed Scopus (48) Google Scholar). In this context, it is interesting to note that the metal binding decreases proton uptake by 2 pH units in response to the formation of Q –A and Q –B symmetrically, as suggested in the same study (15.Gerencser L. Maroti P. Biochemistry. 2001; 40: 1850-1860Crossref PubMed Scopus (48) Google Scholar). Based on spectroscopic measurements, seven titratable/polar residues His-H126, His-H128, Asp-M17, Asp-L210, Glu-L212, Asp-L213, and Ser-L223 were suggested to be involved in PT events of proton uptake at QB (reviewed in Ref. 1.Nabedryk E. Breton J. Okamura M.Y. Paddock M.L. Biochemistry. 2001; 40: 13826-13832Crossref PubMed Scopus (24) Google Scholar). These seven residues are located along three water channels, P1–P3 (reviewed in Ref. 16.Abresch E.C. Paddock M.L. Stowell M.H.B. McPhillips T.M. Axelrod H.L. Soltis S.M. Rees D.C. Okamura M.Y. Feher G. Photosynth. Res. 1998; 55: 119-125Crossref Google Scholar), found in the crystal structure of bRC at 2.2-Å resolution (9.Stowell M.H.B. McPhillips T.M. Rees D.C. Solitis S.M. Abresch E. Feher E. Science. 1997; 276: 812-816Crossref PubMed Scopus (730) Google Scholar). Two of these water channels, P1 and P2 (9.Stowell M.H.B. McPhillips T.M. Rees D.C. Solitis S.M. Abresch E. Feher E. Science. 1997; 276: 812-816Crossref PubMed Scopus (730) Google Scholar), were proposed to connect QB with the solvent on the cytoplasm side and are considered to participate in proton uptake at QB. Water channel P1 extends about 23 Å from QB via Glu-L212, Lys-H130, Glu-H122, Glu-M236 to Arg-H70, and His-H68 at the cytoplasmic side in an approximately orthogonal orientation to the membrane surface. It connects QB mainly through subunit H with Asp-H224 (P1a) or Asp-M240 (P1b) as surface residue. Water channel P2, which is oriented essentially parallel to the membrane surface, is 20 Å, slightly shorter than P1. It connects QB via Ser-L223, Asp-L213, Asn-M44, Glu-H173, and Gln-H174 with the surface residue Gln-M11 or Tyr-M3 (9.Stowell M.H.B. McPhillips T.M. Rees D.C. Solitis S.M. Abresch E. Feher E. Science. 1997; 276: 812-816Crossref PubMed Scopus (730) Google Scholar, 16.Abresch E.C. Paddock M.L. Stowell M.H.B. McPhillips T.M. Axelrod H.L. Soltis S.M. Rees D.C. Okamura M.Y. Feher G. Photosynth. Res. 1998; 55: 119-125Crossref Google Scholar). Water channel P3, proposed more recently, connects QB via Asp-L213 with the surface-exposed Asp-M17 (16.Abresch E.C. Paddock M.L. Stowell M.H.B. McPhillips T.M. Axelrod H.L. Soltis S.M. Rees D.C. Okamura M.Y. Feher G. Photosynth. Res. 1998; 55: 119-125Crossref Google Scholar). Since there exists a patch of closely interacting acidic residues (Asp-L210, Asp-L213, Asp-M17, Asp-H124, Asp-H170, and Glu-H173) and an extended water cluster in the neighborhood, the water channels can be partially delocalized (16.Abresch E.C. Paddock M.L. Stowell M.H.B. McPhillips T.M. Axelrod H.L. Soltis S.M. Rees D.C. Okamura M.Y. Feher G. Photosynth. Res. 1998; 55: 119-125Crossref Google Scholar). In the present study, we report the changes in protonation pattern and pKa values of titratable residues with respect to the wild type bRC, focusing on the Q 0AQ –B state for inhibited (D(L213)N single mutant) and revertant (D(L213)A/R(M233)C or D(L213)A/R(H177)H double mutant) mutant bRC (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The double mutant Glu-L212/Asp-L213 to Ala (AA mutant) is known to interrupt the ET-coupled PT reaction after the first ET event at QB (6.Hanson D.K. Baciou L. Tiede D.M. Nance S.L. Schiffer M. Sebban P. Biochim. Biophys. Acta. 1992; 1102: 260-265Crossref PubMed Scopus (60) Google Scholar, 17.Maróti P. Hanson D.K. Schiffer M. Sebban P. Nat. Struct. Biol. 1995; 2: 1057-1059Crossref PubMed Scopus (54) Google Scholar). To elucidate the PT pathway in bRC, we also investigated the AA mutant bRC, and the results were compared with those calculated for the revertant or wild type bRC. Atomic Coordinates and Charges—In our computations, we used crystal structures of the bRC from Rhodobacter sphaeroides for the D(L213)N single mutant with QB at the proximal position (Protein Data Bank code 1RY5), the D(L213)N/R(M233)C double mutant in the P+QB– state (Protein Data Bank code 1S00), the D(L213)N/R(H177)H double mutant (Protein Data Bank code 1RVJ) (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), the E(L212)A/D(L213)A double mutant (AA mutant) (Protein Data Bank code 1K6N) (17.Maróti P. Hanson D.K. Schiffer M. Sebban P. Nat. Struct. Biol. 1995; 2: 1057-1059Crossref PubMed Scopus (54) Google Scholar), and the wild type light-exposed structure (Protein Data Bank code 1AIG) (9.Stowell M.H.B. McPhillips T.M. Rees D.C. Solitis S.M. Abresch E. Feher E. Science. 1997; 276: 812-816Crossref PubMed Scopus (730) Google Scholar) in the P+Q –B state. The atomic coordinates were prepared in the same way as in previous applications (18.Ishikita H. Morra G. Knapp E.W. Biochemistry. 2003; 42: 3882-3892Crossref PubMed Scopus (53) Google Scholar, 19.Ishikita H. Knapp E.W. J. Am. Chem. Soc. 2004; 126: 8059-8064Crossref PubMed Scopus (52) Google Scholar, 20.Ishikita H. Loll B. Biesiadka J. Galstyan A. Saenger W. Knapp E.W. FEBS Lett. 2005; 579: 712-716Crossref PubMed Scopus (17) Google Scholar). The position of hydrogen atoms were energetically optimized with CHARMM (21.Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comp. Chem. 1983; 4: 187-217Crossref Scopus (14019) Google Scholar) using the CHARMM22 force field. During this procedure, the positions of all nonhydrogen atoms were fixed, and all titratable groups were kept in their standard charge state (i.e. basic groups were considered to be protonated, and acidic groups were considered to be ionized). All of the other atoms whose coordinates were available in the crystal structure were not geometry optimized. Atomic partial charges of the amino acids were adopted from the all-atom CHARMM22 (21.Brooks B.R. Bruccoleri R.E. Olafson B.D. States D.J. Swaminathan S. Karplus M. J. Comp. Chem. 1983; 4: 187-217Crossref Scopus (14019) Google Scholar) parameter set. For cofactors and residues whose charges are not available in CHARMM22, we used atomic partial charges from previous applications (18.Ishikita H. Morra G. Knapp E.W. Biochemistry. 2003; 42: 3882-3892Crossref PubMed Scopus (53) Google Scholar, 19.Ishikita H. Knapp E.W. J. Am. Chem. Soc. 2004; 126: 8059-8064Crossref PubMed Scopus (52) Google Scholar, 20.Ishikita H. Loll B. Biesiadka J. 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The dielectric constant was set to ϵP = 4 inside the protein and ϵW = 80 for water. All computations were performed at 300 K with pH 7.0 and an ionic strength of 100 mm. The linear Poisson Boltzmann equation was solved using a three-step gridfocusing procedure with a starting grid resolution of 2.5 Å, an intermediate grid resolution of 1.0 Å, and a final grid resolution of 0.3 Å. Monte Carlo sampling yields the probabilities [A–] and [HA] of the deprotonated and protonated state of the titratable residue A, respectively. With the Henderson-Hasselbalch equation, pH=pKa+log[A−][HA] (Eq. 1) the pKa value can be calculated as the pH where the concentration of [A–] and [HA] are equal. The protonation patterns were computed for the QA0QB– redox state if not otherwise stated. The procedures to obtain pKa of titratable residues are equivalent to those of the redox potential for redox-active groups, although in the latter case the Nernst equation is applied instead of Equation 1 (33.Ullmann G.M. Knapp E.W. Eur. Bophys. J. 1999; 28: 533-551Crossref PubMed Scopus (226) Google Scholar). Therefore, the accuracy of the present pKa computations is directly comparable with that obtained for recent computations on redox-active cofactors in bRC (18.Ishikita H. Morra G. Knapp E.W. Biochemistry. 2003; 42: 3882-3892Crossref PubMed Scopus (53) Google Scholar, 19.Ishikita H. Knapp E.W. J. Am. Chem. Soc. 2004; 126: 8059-8064Crossref PubMed Scopus (52) Google Scholar, 20.Ishikita H. Loll B. Biesiadka J. Galstyan A. Saenger W. Knapp E.W. FEBS Lett. 2005; 579: 712-716Crossref PubMed Scopus (17) Google Scholar), photosystem I (PSI) (34.Ishikita H. Knapp E.W. J. Biol. 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Breton J. Okamura M.Y. Paddock M.L. Biochemistry. 1998; 37: 14457-14462Crossref PubMed Scopus (35) Google Scholar) and our previous computations (19.Ishikita H. Knapp E.W. J. Am. Chem. Soc. 2004; 126: 8059-8064Crossref PubMed Scopus (52) Google Scholar, 23.Rabenstein B. Ullmann G.M. Knapp E.W. Biochemistry. 2000; 39: 10487-10496Crossref PubMed Scopus (103) Google Scholar), Glu-H173 in wild type bRC remains deprotonated regardless of the redox state of QB. The latter fact implies a small pKa for Glu-H173, whereas at the same time, it does not exclude transient protonation that may be required for the PT events at QB. Hence, the protonation state at Glu-H173 in the revertant mutants may suggest also a participation of Glu-H173 in the PT process coupled with formation of Q –B (7.Paddock M.L. Feher G. Okamura M.Y. FEBS Lett. 2003; 555: 45-50Crossref PubMed Scopus (104) Google Scholar, 10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 45.Gunner M.R. Zhu Z. Structure. 2004; 12: 518-519Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar).Table IProtonation probability of residues for different QA/B redox statesResiduesRedox statesWTaWild-type bRC.D(L213)N mutantsSingle mutantcSingle D(L213)N (inhibited) mutant bRC.AA mutantfAA (inhibited) mutant bRC.R(M233)CbRevertant mutant bRC with double mutation.R(H177)HbRevertant mutant bRC with double mutation.AdConformation A of Glu-H173 (Protein Data Bank code 1RY5), where it forms a salt bridge with Arg-H177 (10).BeConformation B of Glu-H173 (Protein Data Bank code 1RY5), where it forms hydrogen bonds with Asn-L213 and Thr-L226 (10).Glu-L212QA0QB00.020.010.030.010.03QA0QB−1.000.870.741.001.00Glu-H173QA0QB00.000.260.020.000.000.00QA0QB−0.000.160.130.000.000.00Glu-M236QA0QB00.990.980.780.580.380.00QA0QB−0.970.970.490.120.060.00a Wild-type bRC.b Revertant mutant bRC with double mutation.c Single D(L213)N (inhibited) mutant bRC.d Conformation A of Glu-H173 (Protein Data Bank code 1RY5), where it forms a salt bridge with Arg-H177 (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).e Conformation B of Glu-H173 (Protein Data Bank code 1RY5), where it forms hydrogen bonds with Asn-L213 and Thr-L226 (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).f AA (inhibited) mutant bRC. Open table in a new tab Interestingly, in the revertant mutants, the computed proton uptake at Glu-L212 upon formation of the QA0QB– state was lower than in the wild type and inhibited mutant bRC (Table I). This reduced proton uptake at Glu-L212 was approximately compensated by additional proton uptake at Glu-H173. The observed proton uptake at Glu-H173 in revertant mutant bRC implies a pKa increase upon formation of the QA0QB– state. Thus, with respect to wild type bRC, we observed a considerable increase of the calculated pKa for Glu-H173 by about 3.5 units in both revertant mutants (Table II). It has been suggested that a rearrangement of the side chains of charged residues like Arg-H177 increases the pKa for Glu-H173 such that it can function equally well as proton donor and acceptor as needed in the PT chain connecting the solvent with QB (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 45.Gunner M.R. Zhu Z. Structure. 2004; 12: 518-519Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Indeed, our computations showed that the pKa for Glu-H173 is larger than 6 in both revertant mutants. Contrary to the revertant mutants, the inhibited mutants and the AA mutants showed a pKa for Glu-H173 that is 1.1 and 3.2 units lower than in the wild type bRC, respectively (Table II).Table IIpKA of acidic residues in the QA0QB− stateResiduesWTaWild-type bRC.D(L213)N mutantsSingle mutantcSingle D(L213)N (inhibited) mutant bRC.AA mutantfAA (inhibited) mutant bRC.R(M233)CbRevertant mutant bRC with double mutation.R(H177)HbRevertant mutant bRC with double mutation.AdConformation A of Glu-H173 (Protein Data Bank code 1RY5), where it forms a salt bridge with Arg-H177 (10).BeConformation B of Glu-H173 (Protein Data Bank code 1RY5), where it forms hydrogen bonds with Asn-L213 and Thr-L226 (10).H (inside) Glu-H1221.54.32.41.61.6-0.7 Asp-H170-4.71.10.7-5.6-5.6-4.2 Glu-H1732.76.36.21.61.4-0.5 Glu-H2300.95.53.01.11.1-2.0H (surface) Asp-H1194.62.93.43.03.02.6 Glu-H2243.83.34.24.24.23.6 Glu-H2294.34.16.56.56.54.1L Asp-L2103.03.01.92.72.62.0 Glu-L2129.47.87.510.410.6 Asp-L2138.9M Asp-M175.43.53.84.04.23.0 Glu-M232-0.71.45.02.81.2-2.1 Glu-M2368.48.57.06.15.84.7a Wild-type bRC.b Revertant mutant bRC with double mutation.c Single D(L213)N (inhibited) mutant bRC.d Conformation A of Glu-H173 (Protein Data Bank code 1RY5), where it forms a salt bridge with Arg-H177 (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).e Conformation B of Glu-H173 (Protein Data Bank code 1RY5), where it forms hydrogen bonds with Asn-L213 and Thr-L226 (10.Xu Q. Axelrod H.L. Abresch E.C. Paddock M.L. Okamura M.Y. Feher G. Structure. 2004; 12: 703-715Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar).f AA (inhibited) mutant bRC. Open table in a new tab In the wild type bRC, the calculated pKa of 5.4 for Asp-M17 is significantly larger than that of 2.7 for Glu-H173, which favors an involvement of Asp-M17 rather than Glu-H173 in the PT pathway (44.Nabedryk E. Breton J. Okamura M.Y. Paddock M.L. Biochemistry. 1998; 37: 14457-14462Crossref PubMed Scopus (35) Google Scholar, 46.Paddock M.L. Adelroth P. Chang C. Abresch E.C. Feher G. Okamura M.Y. Biochemistry. 2001; 40: 6893-6902Crossref PubMed Scopus (44) Google Scholar). Notably, all mutant bRC considered here showed a pKa decrease for Asp-M17 by 1.4–2.4 units with respect to wild type bRC (Table II). This is predominantly due to Asp-L213 that is mutated to a nontitratable residue of vanishing total charge. Since only the revertant mutant bRCs have a significantly larger pKa of 6.2–6.3 for Glu-H173 as compared with the pKa of 3.5–3.8 for Asp-M17, it is unlikely that the other
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