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 thermodynamic characteristic of four-heme cytochrome c in Rhodopseudomonas viridis reaction centers, as derived from a quantitative analysis of the differential absorption spectra in a-domain* A method of decomposing of the absorption spectrum of four-heme cytochrome of a Rhodopseudomonus viridis reaction center preparation into spectra of individual components was used to estimate the degree of the reduction of hemes as a function of redox potential in the medium. The method enables an evaluation of the shape of redox-titration curves of each heme. The redox-titration curves derived by this approach are approximated well by a Nemst equation with n= 1 and &,-values of 360 mV, 312 mV, 20 mV and less than -50 mV. For all of the redox species the values of midpoint potential estimates by the above method are in good agreement with those determined earlier using another procedure [Dracheva et al. (1988) Eur. J. Biochem. 171, 253-2641. The accuracy of deconvolution of data is within the experimental errors of the redox potential measurement.
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