Abstract
Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asp-96-->Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96-->Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96-->Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.
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- Braiman M. S., Mogi T., Marti T., Stern L. J., Khorana H. G., Rothschild K. J. Vibrational spectroscopy of bacteriorhodopsin mutants: light-driven proton transport involves protonation changes of aspartic acid residues 85, 96, and 212. Biochemistry. 1988 Nov 15;27(23):8516–8520. doi: 10.1021/bi00423a002. [DOI] [PubMed] [Google Scholar]
- Braiman M., Mathies R. Resonance Raman evidence for an all-trans to 13-cis isomerization in the proton-pumping cycle of bacteriorhodopsin. Biochemistry. 1980 Nov 11;19(23):5421–5428. doi: 10.1021/bi00564a042. [DOI] [PubMed] [Google Scholar]
- Butt H. J., Fendler K., Bamberg E., Tittor J., Oesterhelt D. Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump. EMBO J. 1989 Jun;8(6):1657–1663. doi: 10.1002/j.1460-2075.1989.tb03556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Danshina S. V., Drachev L. A., Kaulen A. D., Korana Kh G., Marti T., Mogi T., Skulachev V. I. Issledovanie intermediatea N s pomoshch'iu mutantnykh form bakteriorhodopsina po Asp-96. Biokhimiia. 1992 Oct;57(10):1574–1585. [PubMed] [Google Scholar]
- Engelhard M., Gerwert K., Hess B., Kreutz W., Siebert F. Light-driven protonation changes of internal aspartic acids of bacteriorhodopsin: an investigation by static and time-resolved infrared difference spectroscopy using [4-13C]aspartic acid labeled purple membrane. Biochemistry. 1985 Jan 15;24(2):400–407. doi: 10.1021/bi00323a024. [DOI] [PubMed] [Google Scholar]
- Gerwert K., Hess B., Soppa J., Oesterhelt D. Role of aspartate-96 in proton translocation by bacteriorhodopsin. Proc Natl Acad Sci U S A. 1989 Jul;86(13):4943–4947. doi: 10.1073/pnas.86.13.4943. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gerwert K., Souvignier G., Hess B. Simultaneous monitoring of light-induced changes in protein side-group protonation, chromophore isomerization, and backbone motion of bacteriorhodopsin by time-resolved Fourier-transform infrared spectroscopy. Proc Natl Acad Sci U S A. 1990 Dec 15;87(24):9774–9778. doi: 10.1073/pnas.87.24.9774. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heberle J., Dencher N. A. Bacteriorhodopsin in ice. Accelerated proton transfer from the purple membrane surface. FEBS Lett. 1990 Dec 17;277(1-2):277–280. doi: 10.1016/0014-5793(90)80864-f. [DOI] [PubMed] [Google Scholar]
- Hegemann P., Oesterbelt D., Steiner M. The photocycle of the chloride pump halorhodopsin. I: Azide-catalyzed deprotonation of the chromophore is a side reaction of photocycle intermediates inactivating the pump. EMBO J. 1985 Sep;4(9):2347–2350. doi: 10.1002/j.1460-2075.1985.tb03937.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
- Hessling B., Souvignier G., Gerwert K. A model-independent approach to assigning bacteriorhodopsin's intramolecular reactions to photocycle intermediates. Biophys J. 1993 Nov;65(5):1929–1941. doi: 10.1016/S0006-3495(93)81264-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holz M., Drachev L. A., Mogi T., Otto H., Kaulen A. D., Heyn M. P., Skulachev V. P., Khorana H. G. Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2167–2171. doi: 10.1073/pnas.86.7.2167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Humphrey W., Logunov I., Schulten K., Sheves M. Molecular dynamics study of bacteriorhodopsin and artificial pigments. Biochemistry. 1994 Mar 29;33(12):3668–3678. doi: 10.1021/bi00178a025. [DOI] [PubMed] [Google Scholar]
- Lanyi J. K. Proton translocation mechanism and energetics in the light-driven pump bacteriorhodopsin. Biochim Biophys Acta. 1993 Dec 7;1183(2):241–261. doi: 10.1016/0005-2728(93)90226-6. [DOI] [PubMed] [Google Scholar]
- Lewis A., Spoonhower J., Bogomolni R. A., Lozier R. H., Stoeckenius W. Tunable laser resonance raman spectroscopy of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4462–4466. doi: 10.1073/pnas.71.11.4462. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Longstaff C., Rando R. R. Deprotonation of the Schiff base of bacteriorhodopsin is obligate in light-induced proton pumping. Biochemistry. 1987 Sep 22;26(19):6107–6113. doi: 10.1021/bi00393a024. [DOI] [PubMed] [Google Scholar]
- Maeda A., Sasaki J., Shichida Y., Yoshizawa T. Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy. Biochemistry. 1992 Jan 21;31(2):462–467. doi: 10.1021/bi00117a023. [DOI] [PubMed] [Google Scholar]
- Nagle J. F., Tristram-Nagle S. Hydrogen bonded chain mechanisms for proton conduction and proton pumping. J Membr Biol. 1983;74(1):1–14. doi: 10.1007/BF01870590. [DOI] [PubMed] [Google Scholar]
- Oesterhelt D., Stoeckenius W. Functions of a new photoreceptor membrane. Proc Natl Acad Sci U S A. 1973 Oct;70(10):2853–2857. doi: 10.1073/pnas.70.10.2853. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Oesterhelt D., Stoeckenius W. Isolation of the cell membrane of Halobacterium halobium and its fractionation into red and purple membrane. Methods Enzymol. 1974;31:667–678. doi: 10.1016/0076-6879(74)31072-5. [DOI] [PubMed] [Google Scholar]
- Otto H., Marti T., Holz M., Mogi T., Lindau M., Khorana H. G., Heyn M. P. Aspartic acid-96 is the internal proton donor in the reprotonation of the Schiff base of bacteriorhodopsin. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9228–9232. doi: 10.1073/pnas.86.23.9228. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Papadopoulos G., Dencher N. A., Zaccai G., Büldt G. Water molecules and exchangeable hydrogen ions at the active centre of bacteriorhodopsin localized by neutron diffraction. Elements of the proton pathway? J Mol Biol. 1990 Jul 5;214(1):15–19. doi: 10.1016/0022-2836(90)90140-h. [DOI] [PubMed] [Google Scholar]
- Takahashi E., Wraight C. A. Small weak acids stimulate proton transfer events in site-directed mutants of the two ionizable residues, GluL212 and AspL213, in the QB-binding site of Rhodobacter sphaeroides reaction center. FEBS Lett. 1991 May 20;283(1):140–144. doi: 10.1016/0014-5793(91)80572-k. [DOI] [PubMed] [Google Scholar]
- Tittor J., Soell C., Oesterhelt D., Butt H. J., Bamberg E. A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide. EMBO J. 1989 Nov;8(11):3477–3482. doi: 10.1002/j.1460-2075.1989.tb08512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]