The mechanism of H<sup>+</sup> transfer by bacteriorhodopsin The properties and the function of intermediate P

In our previous work [(1993) FEBS Lett. 313,248-250; Biochem. Int. 30,[461][462][463][464][465][466][467][468][469] M-intermediate formation of wild-type bacteriorhodopsin was shown to involve two components differing in time constants (z, = 6&7Ops and z2 = 22&25Ops), which were suggested to reflect two independent pathways of M-intermediate formation. The contribution of the fast M was 4-times higher than the slow one. Our present research on M-intermediate formation in the Dll5N bacteriorhodopsin mutant revealed the same components but at a contribution ratio of 1:l. Upon lowering the pH, the slow phase of M-formation vanished at a pK of 6.2, and in the pH region 3.G5.5 only the M-intermediate with a rise time of 60 pus was present. A 5-6 h incubation of D115N bacteriorhodopsin at pH 10.6 resulted in the irreversible transformation of 50% of the protein into a form with a difference absorbance maximum at 460 nm. This form was stable at pH 7.5 and had no photocycle, including M-intermediate formation. The remaining bactenorhodopsin contained 100% fast M-intermediate. The disappearance of the 250;~s phase concomitant with bR460 formation indicates that at neutral pH bacteriorhodopsin exists as two spectroscopically indistinguishable forms.

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“…The pm preparation, photocycle registration and absorbance spectra measurements were described elsewhere [1, 2,5].…”

Section: Methodsmentioning

confidence: 99%

On the two forms of bacteriorhodopsin

Komrakov

Kaulen

1994

FEBS Letters

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“…The methods for preparing the purple membrane (pro), for measuring photocycle intermediates and pH changes were described elsewhere [5]. PyranJne was employed as a pl-I indicator.…”

Section: Methodsmentioning

confidence: 99%

Interrelations of M‐intermediates in bacteriorhodopsin photocycle

1992

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The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by tile flash-photolysis technique. Tile M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, rt = 65 and r, = 250/~s) for the wild-type bR and thr~ exponents (r~ = 55 ,us, r,. = 220 as and r.a = 1 ms) for the D96N mutant of bR. A component with r = 1 ms was found to be prescott in the photoeycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu ~* ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the I-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the l-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp~-~Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identi~l for th~ wild-type bR and D96N mutant and matched the M->M' eonlbrmational transition, A model for M rise in the bR photocycle is proposed.

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“…It is also associated with the transmembrane pumping of at least one, or probably two, protons (3,23). Vectorial movements of these protons within the membrane-incorporated enzyme result in the generation of a transmembrane electric potential difference (⌬⌿) that can be monitored electrometrically with submicrosecond time resolution using the method developed by Drachev (24)(25)(26).…”

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confidence: 99%

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

Konstantinov

Siletsky

Mitchell

et al. 1997

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

346

393

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The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a 3 (Fe 4؉ ‫؍‬ O 2؊ ) to the oxidized form (Fe 3؉ OH ؊ ). This redox step requires the delivery of a ''chemical'' H ؉ to protonate the reduced oxygen atom and is also coupled to proton pumping. It is found that mutations in the K channel (K362M and T359A) have virtually no effect on the ferryl-oxo-to-oxidized (F-to-Ox) transition, although steady-state turnover is severely limited. In contrast, electrogenic proton transfer at this step is strongly

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The mechanism of H⁺ transfer by bacteriorhodopsin The properties and the function of intermediate P

Cited by 47 publications

References 7 publications

On the two forms of bacteriorhodopsin

On the two forms of bacteriorhodopsin

Interrelations of M‐intermediates in bacteriorhodopsin photocycle

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

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The mechanism of H+ transfer by bacteriorhodopsin The properties and the function of intermediate P

Cited by 47 publications

References 7 publications

On the two forms of bacteriorhodopsin

On the two forms of bacteriorhodopsin

Interrelations of M‐intermediates in bacteriorhodopsin photocycle

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer

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The mechanism of H⁺ transfer by bacteriorhodopsin The properties and the function of intermediate P