Protonation of a novel intermediate P is involved in the M → bR step of the bacteriorhodopsin photocycle

AbstractA novel intermediate (P) of the bacteriorhodopsin (bR) photocycle, appearing between M412 and bR is described. Like bR, intermediate P shows an absorption maximum at 560–570 nm. However, the extinction coefficient of P is somewhat lower than that of bR. Moreover, there are some differences in spectra of bR and P at wavelengths shorter than 450 nm. The P → bR transition correlates with the absorption of H+ from the water medium. The following conditions proved to be favourable for the detection of the new intermediate: a high salt concentration, low light intensity and low temperature (0.5°C). The P → bR transition is strongly decelerated by a small amount of Triton X-100. Illumination of P does not produce M412 before bR is formed. It is assumed that M412 converts to P when the Schiff base is protonated by a proton transferred from a protein protolytic group which participates in the inward H+-conductivity pathway. Reprotonation of this group results in the conversion of P to bR. No more than 1 H+ is transported per bR photocycle

Editorial vol. 3

Direct measurement of the electrogenic activity of purified mitochondrial transhydrogenase has been carried out. To this end, beef‐heart transhydrogenase was isolated and reconstituted with phospholipids to form proteoliposomes. The transhydrogenase proteoliposomes were incorporated into a membrane filter impregnated with a decane solution of phospholipids. It is shown that addition of substrates of either the forward (NADPH and NAD+) or the reverse (NADH and NADP+) transhydrogenase reaction gives rise to an electric potential difference across the proteoliposometreated membrane filter. The electric vector depends upon the direction of the reaction. The proteoliposome‐supplemented compartment charges negatively in the case of the forward reaction and positively in the case of the reverse one. Addition of the reaction products after substrates equalizes the potentials. The transhydrogenase‐treated membrane filter retains the ability to perform transhydrogenase‐linked electrogenesis after removal of excess non‐incorporated proteoliposomes. The electric potential difference reaching 20 mV immediately after the transhydrogenase substrate addition, slowly decreases due to accumulation of the reaction products. Such decay is prevented when the mixture is supplemented with the substrate‐regenerating and product‐utilizing enzymic systems. Under these conditions, a steady continuous electric current of about 10 pA can be observed. Copyright © 1980, Wiley Blackwell. All rights reserve

Reconstitution of Biological Molecular Generators of Electric Current: Transhydrogenase

Direct measurement of the electrogenic activity of purified mitochondrial transhydrogenase has been carried out. To this end, beef‐heart transhydrogenase was isolated and reconstituted with phospholipids to form proteoliposomes. The transhydrogenase proteoliposomes were incorporated into a membrane filter impregnated with a decane solution of phospholipids. It is shown that addition of substrates of either the forward (NADPH and NAD+) or the reverse (NADH and NADP+) transhydrogenase reaction gives rise to an electric potential difference across the proteoliposometreated membrane filter. The electric vector depends upon the direction of the reaction. The proteoliposome‐supplemented compartment charges negatively in the case of the forward reaction and positively in the case of the reverse one. Addition of the reaction products after substrates equalizes the potentials. The transhydrogenase‐treated membrane filter retains the ability to perform transhydrogenase‐linked electrogenesis after removal of excess non‐incorporated proteoliposomes. The electric potential difference reaching 20 mV immediately after the transhydrogenase substrate addition, slowly decreases due to accumulation of the reaction products. Such decay is prevented when the mixture is supplemented with the substrate‐regenerating and product‐utilizing enzymic systems. Under these conditions, a steady continuous electric current of about 10 pA can be observed. Copyright © 1980, Wiley Blackwell. All rights reserve