Abstract
The light-driven proton pump bacteriorhodopsin (BR) from Halobacterium salinarum is tightly regulated by the [H+] gradient and transmembrane potential. BR exhibits optoelectric properties, since spectral changes during the photocycle are kinetically controlled by voltage, which predestines BR for optical storage or processing devices. BR mutants with prolonged lifetime of the blue-shifted M intermediate would be advantageous, but the optoelectric properties of such mutants are still elusive. Using expression in Xenopus oocytes and two-electrode voltage-clamping, we analyzed photocurrents of BR mutants with kinetically destabilized (F171C, F219L) or stabilized (D96N, D96G) M intermediate in response to green light (to probe H+ pumping) and blue laser flashes (to probe accumulation/decay of M). These mutants have divergent M lifetimes. As for BR-WT, this strictly correlates with the voltage dependence of H+ pumping. BR-F171C and BR-F219L showed photocurrents similar to BR-WT. Yet, BR-F171C showed a weaker voltage dependence of proton pumping. For both mutants, blue laser flashes applied during and after green-light illumination showed reduced M accumulation and shorter M lifetime. In contrast, BR-D96G and BR-D96N exhibited small photocurrents, with nonlinear current-voltage curves, which increased strongly in the presence of azide. Blue laser flashes showed heavy M accumulation and prolonged M lifetime, which accounts for the strongly reduced H+ pumping rate. Hyperpolarizing potentials augmented these effects. The combination of M-stabilizing and -destabilizing mutations in BR-D96G/F171C/F219L (BR-tri) shows that disruption of the primary proton donor Asp-96 is fatal for BR as a proton pump. Mechanistically, M destabilizing mutations cannot compensate for the disruption of Asp-96. Accordingly, BR-tri and BR-D96G photocurrents were similar. However, BR-tri showed negative blue laser flash-induced currents even without actinic green light, indicating that Schiff base deprotonation in BR-tri exists in the dark, in line with previous spectroscopic investigations. Thus, M-stabilizing mutations, including the triple mutation, drastically interfere with electrochemical H+ gradient generation.
Highlights
Bacteriorhodopsin (BR) from Halobacterium salinarum acts as a light-driven proton pump
As observed for BR-WT (Fig. 2A), all investigated mutants respond to green light (l .495 nm) with positive transient and stationary photocurrents consistent with outwardly directed H+ translocation (Fig. 3A,E; Fig. 4A,F; and Fig. 5A) indicating correct right-side-out insertion in the cellular membrane
The peak amplitudes are less voltage-dependent than the stationary currents, which can be attributed to a lower voltage dependence of the initial photocycle events
Summary
Bacteriorhodopsin (BR) from Halobacterium salinarum acts as a light-driven proton pump. It generates an electrochemical gradient for protons, which is utilized for secondary active transport processes and ATP synthesis. The key steps in the BR photocycle for the directed, ‘vectorial’ transport process are the de- and reprotonation of the Schiff base (i.e. the formation of the M1 intermediate, the transition from M1 to the intermediate M2, and decay of M2). During the formation of M1, the Schiff base (SB) releases a proton to Asp-85, and its pKa shifts from .13 to ,4. The retinal SB is reprotonated by Asp-96 from the intracellular side, whereby M2 relaxes back to the ground state BR via the photocycle intermediates N and O. The MRBR decay contains the rate-limiting and main electrogenic steps at depolarizing voltages [1]
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