The photochemical cycle and the proton-pumping function of bacteriorhodopsin modified with lanthanum and formaldehyde has been studied. In both preparations, the M412 + BR570 transition time has been found to increase considerably. The deceleration of the photochemical cycle has been shown to be accompanied by inhibition of the millisecond phase of the photoelectrical response of bacteriorhodopsin membranes associated with phospholipidimpregnated collodion film. Photoelectrogenic activity measured with permeable ion probe in proteoliposomes was also inhibited. Effects of lanthanum were reversed by EDTA. Formation of M412 was slightly accelerated and the microsecond electrogenic phase was not affected by lanthanum and by formaldehyde. It is concluded that lanthanum, but not formaldehyde, can be used as a specific reversible inhibitor of the second half of the bacteriorhodopsin photocycle and of the associated H + uptake on the cytoplasmic side of the halobacterial membrane. Possible mechanisms of these effects are discussedBacteriorhodopsin is a bacterial proton pump that utilizes the energy of light. The properties of this protein have been investigated since 1971 [l, 21 by a variety physical and chemical methods (reviews [ 3 , 41). Its primary structure has been determined [5]. However, the mechanism of the translocation of the H+-ion remains to be elucidated.Having absorbed light, the bacteriorhodopsin molecule undergoes a cycle of photochemical conversions as a result of which H + ion(s) is(are) released on the outer side of the plasma membrane and bound on its cytoplasmic side. The central intermediate of the photochemical cycle with the absorption maximum at 412 (M412) does not have, in contrast to the initial state of bacteriorhodopsin, a proton at the Schiff base formed by retinal and E-NH,-group of the 216th lysine. The electrogenic phases of the bacteriorhodopsin cycle are revealed on pulse excitation of a planar artificial film with purple membranes adsorbed on it [6-lo]. We have established the existence of the following phases in the electric response to a light flash: (1) a negative phase; (2) a microsecond phase; (3) a millisecond phase; (4) relaxation of the potential difference that is due to the membrane capacity discharge. Phases 2 and 3 have the kinetics that is similar to that of the formation and relaxation of M412. The absence of the strict conformity between the rates of the spectral and electrical events [9, 101 testifies to the complexity of the transient states of the molecule. As it was found previously lanthanum ions inhibit the millisecond phase of the photoelectric response [6, 7, 10, 1 I] as well as the relaxation of M412 [lo, 121. The modification of purple membranes by formaldehyde [13, 141 also results in deceleration of the photochemical cycle.In this paper we have studied the deceleration of the photochemical cycle by lanthanum ions and formaldehydeAbbreviations. Mes, 4-morpholineethanesulfonic acid; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; PCB-, phenyldicarbau...
The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.
Bacteriorhodopsin‐containing fragments of Halobacterium halobium membrane (bacteriorhodopsin sheets) were incorporated into a lecithin‐impregnated collodion film, and fast stages of flash‐induced electrogenesis were measured by two electrodes separated by this film. It is found that a single turnover of bacteriorhodopsin results in an electrogenic response composed of three main stages of the following τ: the first < 200 ns, the second 15–70 μs and the third 10 ms. The second and third phases are of the same direction as an electric response to continuous illumination, whereas the first one is oppositely directed. The lts and ms stages were shown to correlate, in the first approximation, with formation and decomposition of the bacteriorhodopsin intermediate absorbing at 412 nm, respectively. Both the second and third phases of the photoelectric response are sums of at least two exponents. The third stage is specifically inhibition by La3+ ions which are also shown to decrease the rate of regeneration of the original bacteriorhodopsin absorbing at 570 nm from the intermediate absorbing at 412 nm. Acidification of the medium induces parallel inhibition of the second and third phases and of formation of the intermediate absorbing at 412 nm as if protonation of a group with pK= 3.6 were responsible for this inhibition. The first (opposite) phase survives acidification. It even increases at pH lower than 1.5. At such a low pH, one can show a good correlation of decays of photopotential and of a bacteriorhodopsin bathointermediate. The decays are biphasic (τ1= 200 μs and τ2= 2 ms), formation of both the photopotential and the bathointermediate being faster than 200 ns. At higher pH, when a three‐phase photoelectric response is revealed, decay of the formed electric potential difference gives the average τ value of about 1 s. It can be accelerated by compounds that increase ionic conductance of biomembranes. At pH below 4, fluoride is found to completely inhibit the second and third phases, so that only the first phase is observed. The results are discussed in terms of a scheme postulating that the first electrogenic phase is a result of translocation of the protonated Schiff base inside the membrane due to a light‐induced conformation change in retinal or protein. The second and third phases are explained by H+ transfer from the Schiff base to the outer membrane surface and from inner (cytoplasmic) surface of membrane to the Schiff base, respectively.
Photoelectric activity and photocycle kinetics of bacteriorhodopsin successively treated with a carbodiimide and several amine derivatives have been studied. Both spectral and electrical responses were initiated by a 15-1-1s laser flash inducing a single turnover of bacteriorhodopsin. Photocycle kinetics were measured in a suspension of purple sheets. Photoelectric potential generation was monitored by a voltmeter, using a phospholipid-impregnated collodion film covered on one side with purple sheets or proteoliposomes.It is shown that in bacteriorhodopsin treated with a carbodiimide derivative and ethylenediamine or methyl-oarginine, significant acceleration of the millisecond phase of the photoelectric response is observed, whereas the kinetics of the microsecond phase remain unchanged. This effect is accompanied by an increase in the fast (T 2 ms) component of the 41 2-nm intermediate decay. Methylamine-modified bacteriorhodopsin shows no such changes in electric and spectral kinetics.Treatment with ethylenediamine or arginine, rather than methylamine, desensibilizes, completely or partially, photoelectric response to inhibition by cationic effectors such as La3+ or by increase in the H + concentration. At the same time modification sensibilizes this response to the inhibiting effect of an anion, citrate.Dark adaptation of bacteriorhodopsin modified by ethylenediamine or arginine slows down both microsecond and millisecond electrogenic phases, whereas that of untreated or methylamine modified bacteriorhodopsin does not.Proteoliposomes of ethylenediamine-modified bacteriorhodopsin generate a photoelectric potential of opposite direction (interior negative) as compared with those of unmodified or methylamine-modified bacteriorhodopsin.It is concluded that the modifications by ethylenediamine and arginine change the decomposition of the 412-nm intermediate in such a fashion that a portion of this intermediate decays faster. This results in accelerating those electrogenic phases which are associated with the 412-nm intermediate decay. Such an effect may be due to replacement of negatively charged groups in the bacteriorhodopsin polypeptide chain by positively charged ones.Different methods for measurement of changes in the bacteriorhodopsin activity are discussed. The conclusion is made that the analysis of the kinetics of photoelectric potential generation in the purple sheet-collodion film system is a sensitive probe for changes in bacteriorhodopsin activity.The problem of the mechanism of generation of ADH by bacteriorhodopsin is one of the key questions of the modern bioenergetics and membranology. In fact, there are already necessary prerequisites to elucidate how this simplest membrane-linked energy transducer utilizes light energy to carry out the uphill pumping of the H + ions across a halobacterial membrane. The amino acid sequence of bacteriorhodopsin has been known since 1978 [I] (see also [2-41). In 1975, its three-dimensional structure in native membrane was described i.e. much faster than a single...
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