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Role of Arg82 in the early steps of the bacteriorhodopsin proton-pumping cycle.

ABSTRACT: Proton-transfer reactions in the bacteriorhodopsin light-driven proton pump are coupled with structural rearrangements of protein amino acids and internal water molecules. It is generally thought that the first proton-transfer step from retinal Schiff base to the nearby Asp85 is coupled with movement of the Arg82 side chain away from Asp85 and toward the extracellular proton release group. This movement of Arg82 likely triggers the release of the proton from the proton release group to the extracellular bulk. The exact timing of the movement of Arg82 and how this movement is coupled with proton transfer are still not understood in molecular detail. Here, we address these questions by computing the free energy for the movement of the Arg82 side chain. The calculations indicate that protonation of Asp85 leads to a fast reorientation of the Arg82 side chain toward the extracellular proton release group.

SUBMITTER: Clemens M

PROVIDER: S-EPMC3135100 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Role of Arg82 in the early steps of the bacteriorhodopsin proton-pumping cycle.

Clemens Maike M Phatak Prasad P Cui Qiang Q Bondar Ana-Nicoleta AN Elstner Marcus M

The journal of physical chemistry. B 20110511 21

Proton-transfer reactions in the bacteriorhodopsin light-driven proton pump are coupled with structural rearrangements of protein amino acids and internal water molecules. It is generally thought that the first proton-transfer step from retinal Schiff base to the nearby Asp85 is coupled with movement of the Arg82 side chain away from Asp85 and toward the extracellular proton release group. This movement of Arg82 likely triggers the release of the proton from the proton release group to the extra ...[more]

PMID: 21561116

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Project description: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.

Project description:The bacterial lifestyle is plastic, requiring transcriptional, translational, and metabolic tailoring for survival. These dynamic cellular processes are energy intensive; therefore, flexible energetics is requisite for adaptive plasticity. An intricate network of complementary and supplementary pathways exists in bacterial energy metabolism. There are two main entry points for electrons in the aerobic electron transport system, NADH dehydrogenase (NDH) and succinate dehydrogenase (SDH), receiving electrons from NADH and succinate, respectively. Aerobic bacterial phyla have a non-proton-pumping NADH dehydrogenase, which is often the primary dehydrogenase under aerobiosis. Here, we report adaptive changes supporting growth restoration in an Escherichia coli strain lacking the primary dehydrogenase. Growth optimization is achieved by reducing the activity of succinate dehydrogenase, and thus we demonstrate a physiological discord between proton-pumping NADH dehydrogenase and succinate dehydrogenase in supporting growth. Beyond the fundamental understanding of the bioenergetic network, identifying this compensatory feature provides impetus to rational antimicrobial combinations for targeting the non-proton-pumping dehydrogenase. IMPORTANCE Energy generation pathways are a potential avenue for the development of novel antibiotics. However, bacteria possess remarkable resilience due to the compensatory pathways, which presents a challenge in this direction. NADH, the primary reducing equivalent, can transfer electrons to two distinct types of NADH dehydrogenases. Type I NADH dehydrogenase is an enzyme complex comprising multiple subunits and can generate proton motive force (PMF). Type II NADH dehydrogenase does not pump protons but plays a crucial role in maintaining the turnover of NAD+. To study the adaptive rewiring of energy metabolism, we evolved an Escherichia coli mutant lacking type II NADH dehydrogenase. We discovered that by modifying the flux through the tricarboxylic acid (TCA) cycle, E. coli could mitigate the growth impairment observed in the absence of type II NADH dehydrogenase. This research provides valuable insights into the intricate mechanisms employed by bacteria to compensate for disruptions in energy metabolism.

Dataset Information

Role of Arg82 in the early steps of the bacteriorhodopsin proton-pumping cycle.

Publications

Role of Arg82 in the early steps of the bacteriorhodopsin proton-pumping cycle.

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