The pK of Schiff base deprotonation in bacteriorhodopsin

Ehrenberg, Benjamin; Lewis, Aaron

doi:10.1016/0006-291x(78)90307-8

Cited by 17 publications

(5 citation statements)

References 8 publications

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“…The proton in such a model would be less dissociable than in water, and this would account for the slower exchange and would give rise to a higher pK than in solution. This is consistent with the result of kinetic resonance Raman spec-.troscopy (5,16) that even at pH 12 the majority of Schiff base linkages are protonated, indicating a pK value 212.…”

Section: -7supporting

confidence: 79%

“…This suggestion arises principally from resonance Raman experiments that demonstrated that the Schiff base is transiently deprotonated (2) [10][11][12][13][14][15][16][17][18][19][20].usec after excitation of the proton pumping cycle (6). In addition, it has been noted in agreement with this suggestion that alterations in the chromophore around the Schiff base seriously alter the proton pumping activity (7).…”

Section: -7supporting

confidence: 57%

See 1 more Smart Citation

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

Ehrenberg¹,

Lewis²,

Porta³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Using rapid mixing techniques and resonance Raman spectroscopy, we have found that the IH/2H exchange time for the Schiff base proton of bacteriorhodopsin in purple membrane is 4.7 msec, when experiments are carried out at pH 2 or pH 7 at room temperature in the dark. We argue that diffusion of neutral water into the membrane is fast on this time scale. Also, model Schiff bases in solution have a pKa between 6 and 7, and we show that such model Schiff bases have much faster exchange rates. Therefore, we conclude that the Schiff base proton in bacteriorhodopsin is protected from interaction with the medium, probably by interaction with a protein group, and this would account for a pKa considerably higher than 6-7.Bacteriorhodopsin is the only protein found in the purple patches that develop in the plasma membrane of Halobacterium halobium when this bacterium is grown under illumination and at low oxygen pressure (1). Bacteriorhodopsin is a chromoprotein composed of retinal complexed to the f-amino group of a lysine residue (1) by a protonated Schiff base (2), and it is well established that the biological role of this membrane protein is to serve as a light-driven proton pump (3, 4). Thus, the purple membrane has generated considerable interest in bioenergetics.It has been suggested (5) that the Schiff base may play an important role in the proton pumping mechanism. This suggestion arises principally from resonance Raman experiments that demonstrated that the Schiff base is transiently deprotonated (2) 10-20.usec after excitation of the proton pumping cycle (6). In addition, it has been noted in agreement with this suggestion that alterations in the chromophore around the Schiff base seriously alter the proton pumping activity (7). Therefore, in this paper we focus on the Schiff base proton by studying its rate of exchange in the dark as a function of the pH of the external medium.For these measurements the purple membrane fragments, which were prepared and purified by the method of Kanner and Racker (8), were suspended in 2H20, and thus the Schiff base was deuterated. This was demonstrated by resonance Raman spectroscopy, in which the 1642 cm-1 C-N+-H stretching mode of bacteriorhodopsin (bR570) shifts to 1621 cm-1 upon suspension in 2H20 (2). The 2H20 suspension was mixed with IH20 by using a continuous-flow rapid mixing technique. The Raman scattering of the mixture was measured at various points downstream, and the disappearance of the 1621 cm-1 C=N+-2H band and the accompanying formation of the 1642 cm-1 C=-N+-H band were monitored. The mixing was performed by using the eight-jet mixer from a Durrum stopped-flow instrument. The calibration of the mixer's dead time was performed in the following way: SoluThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 6571tions of ascorbic acid and K3Fe(CN)6 were mixed in the same apparatus and the kinetic...

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Section: -7supporting

confidence: 79%

Section: -7supporting

confidence: 57%

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

Ehrenberg¹,

Lewis²,

Porta³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…These (21). This last observation leads us to conclude that, for such complex deprotonation and M412 formation to occur at physiological pH and even at pH values as low as 3, a group with pK > 10.2 + 0.3 lacking a proton must in all probability be prepared in the initial events that excite this proton pump.…”

Section: Resultsmentioning

confidence: 97%

“…To obtain the pH dependence of the kinetics of Schiff base deprotonation and M412 production, bacteriorhodopsin suspensions were excited with 50 mW of 457.9 nm laser light and were flowed at various rates through a capillary tube of known diameter, as described (21). The pH of the suspensions was varied and the data were analyzed as discussed in Fig.…”

mentioning

confidence: 99%

Experimental evidence for secondary protein-chromophore interactions at the Schiff base linkage in bacteriorhodopsin: Molecular mechanism for proton pumping

Lewis

Marcus

Ehrenberg

et al. 1978

Proc. Natl. Acad. Sci. U.S.A.

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Resonance Raman spectroscopy of the retinylidene chromophore in various isotopically abled membrane environments together with spectra of isotopically labeled model compounds demonstrates that a secondary protein interaction is present at the protonated Schiff base linkage in bacteriorhodopsin. The data indicate that although the interaction is present in all protonated bacteriorhodopsin species it is absent in unprotonated intermediates. Furthermore, kinetic resonance Raman spectroscopy has been used to monitor the dynamics of Schiff base deprotonation as a function of pH. All our results are consistent with lysine as the interacting group. A structure for the interaction is proposed in which the interacting protein group in an unprotonated configuration is complexed through the Schiff base proton to the Schiff base nitrogen. These data suggest a molecular mechanism for proton pumping and ion gate molecular regulation. In this mechanism, light causes electron redistribution in the retinylidene chromophore, which results in the deprotonation of an amino acid side chain with pK >10.2 ± 0.3 (e.g., arginine). This induces subsequent retinal and protein conformational transitions which eventually lower the pK of the Schiff base complex from >12 before light absorption to 10.2 + 0.3 in microseconds after photon absorption. Finally, in this low pK state the complex can reprotonate the proton-eficient high pK goup generated by light, and the complex is then reprotonated from the opposite side of the membrane.The discovery of the purple membrane in Halobacterium halobium (1) has generated considerable interest in the field of bioenergetics. It has been shown that, when this bacterium is grown under low oxygen tension and in the light, purple membrane patches develop in the plasma membrane (1). These purple membrane patches contain a two-dimensional crystalline array (2) of a single protein that has been called "bacteriorhodopsin" (1) because of its apparent similarities with visual pigments. These similarities include retinal-based chromophores covalently bound via Schiff base linkages to the e-amino group of lysine residues and similar red shifts and subsequent blue shifts in their absorption spectra as a result of chromophore photon absorption. However, unlike visual photoreceptor rhodopsins, which contain the 11-cis isomer of the chromophore and are quantum detectors, bacteriorhodopsin containing the all-trans isomer is an energy converter that acts as a light-driven proton pump (3-5) coupled to ATP synthesis (4, 5) in support of the Mitchell chemosmotic hypothesis (6). Thus, the molecular mechanism underlying-the function of this membrane-bound proton pump is a problem of fundamental importance in biology.In recent years, resonance Raman spectroscopy has become an important tool for monitoring the environmentally and structurally sensitive vibrational spectra of chromophoric sites in macromolecular complexes (7,8 (17). Reconstitutions (18,19) were performed to prepare the following four systems: (i) H-ret +...

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“…Recently some delightful studies have been reported concerning the mode of action of the bacterial purple membrane protein bacteriorhodopsin (Oseroff & Callender, 1974;Marcus & Lewis, 1977;Ehrenberg & Lewis, 1978;Campion et al, 1977a,b;Terner et al, 1979;Smith et al, 1984) and rhodopsin (see below). Bacteriorhodpsin, which is a transmembrane protein, pumps protons consequent upon light absorption in order to produce a gradient for ATP synthesis and possesses a retinal prosthetic group which is attached to the protein by a Schiff's base linkage with a lysine residue.…”

Section: Modern Instrumentationmentioning

confidence: 99%

Infra-red and Raman spectroscopic studies of enzyme structure and function

Wharton¹

1986

Biochemical Journal

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The pK of Schiff base deprotonation in bacteriorhodopsin

Cited by 17 publications

References 8 publications

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

Experimental evidence for secondary protein-chromophore interactions at the Schiff base linkage in bacteriorhodopsin: Molecular mechanism for proton pumping

Infra-red and Raman spectroscopic studies of enzyme structure and function

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