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

Lewis, Aaron; Marcus, Michael A.; Ehrenberg, Benjamin; Crespi, Henry L.

doi:10.1073/pnas.75.10.4642

Cited by 54 publications

(27 citation statements)

References 37 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%

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%

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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“…In one experiment reported in 1981, lysines in BR were substituted with lysines where the ε-amino group was either completely or partial labeled with 15 N [6]. The results disproved the previously suggested [140] hypothesis [18] that two different lysine residues interacted with the retinylidene Schiff base (SB). In a second experiment ε-amino N 15 lysine labeling was combined with proteolytic fragmentation and recombination of intact BR from these fragments [194].…”

Section: Stable Isotope Labeling Of Membrane Proteinsmentioning

confidence: 34%

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Rothschild

2016

BSI

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Abstract. Membrane proteins facilitate some of the most important cellular processes including energy conversion, ion transport and signal transduction. While conventional infrared absorption provides information about membrane protein secondary structure, a major challenge is to develop a dynamic picture of the functioning of membrane proteins at the molecular level. The introduction of FTIR difference spectroscopy around 1980 to study structural changes in membrane proteins along with a number of associated techniques including protein isotope labeling, site-directed mutagenesis, polarization dichroism, attenuated total reflection and time-resolved spectroscopy have led to significant progress towards this goal. It is now possible to routinely detect conformational changes of individual amino acid residues, backbone peptides, binding ligands, chromophores and even internal water molecules under physiological conditions with time-resolution down to nanoseconds. The advent of ultrafast pulsed-IR lasers has pushed this time-resolution down to femtoseconds. The early development of FTIR difference spectroscopy as applied to membrane proteins with special focus on bacteriorhodopsin is reviewed from a personal perspective.

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“…Protein residues that have been suggested as donating or accepting protons from the Schiff base in bacteriorhodopsin include lysine (27,28,31) as well as tyrosine and carboxylate residues (30,32,(35)(36)(37). Our calculations apply directly to the amine group of lysine; although we have not explicitly examined a pairing of a Schiff base with an oxygen base, it is expected that the precepts delineated here would apply with only minor modifications.…”

Section: Case 1: Carbonyl and Hydroxylmentioning

confidence: 99%

“…At the present, it is widely accepted that protonation/deprotonation of the Schiff base linkage is an essential component in the proton pumping activity of bacteriorhodopsin (27)(28)(29)(30)(31)(32)(33)(34). Moreover, light absorption is known to lead to isomerizations about bonds within the retinal chromophore that alter the orientation of the Schiff base with respect to its protein environment (30)(31)(32)(33)(34).…”

Section: Case 1: Carbonyl and Hydroxylmentioning

confidence: 99%

Modification of pK values caused by change in H-bond geometry.

Scheiner¹,

Hillenbrand²

1985

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

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The competition between various groups for a proton is studied by ab initio molecular orbital methods. It is found that reorientations of the two groups involved in a H-bond can reverse the equilibrium position of the proton shared between them. Specifically, the carbonyl and hydroxyl groups were modeled by H2CO and HOH. In the H-bond between these two groups, association of the proton with the carbonyl (H2COH.OH2)+ is favored over the hydroxyl (H2CO-..HOH2)+ when the latter group is situated along a lone pair of the carbonyl oxygen. However, displacement of the water to the C=O axis between the two carbonyl lone pairs reverses the situation and (H2CO HOH2)+ is more stable. A similar reversal of stability is observed in the H-bond involving a Schiff base (modeled by CH2NH) and amine (NH3). In one arrangement where the lone pairs of the two groups point toward one another, the proton prefers the Schiff base to the amine-i.e., (H2CHNH.NH3)+ is more stable than (H2CHN..HNH3)+. On the other hand, rotation of the lone pair of the amine away from the Schiff base nitrogen results in proton transfer across to the amine. These shifts in stability correspond to reversal of relative pK of the groups involved. A fundamental principle emerging from the calculations is that ion-dipole electrostatic interactions favor transfer of a proton to the group that is positioned as closely as possible to the negative end ofthe dipole moment vector ofthe other. The ideas developed here suggest a number of means by which conformational changes may be utilized to shift protons from residue to residue within a protein molecule such as an enzyme or bacteriorhodopsin.

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

Cited by 54 publications

References 37 publications

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

Exchange kinetics of the Schiff base proton in bacteriorhodopsin.

The early development and application of FTIR difference spectroscopy to membrane proteins: A personal perspective

Modification of pK values caused by change in H-bond geometry.

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