Directional Proton Conductance in Bacteriorhodopsin Is Driven by Concentration Gradient, Not Affinity Gradient

Many microorganisms can harvest energy from sun light to establish electrochemical potential across cell membrane by pumping protons outward. Light driven proton pumping against a transmembrane gradient entails exquisite electronic and conformational reconfigurations at fs to ms time scales. However, transient molecular events along the photocycle of bacteriorhodopsin are difficult to comprehend from noisy and inconsistent electron density maps obtained from multiple experiments. A major challenge arises from the coexisting intermediate populations as a heterogenous conformational mixture continuously evolves over 13 decades in time. This study reports a meta-analysis of the recent time-resolved datasets collected by several consortia. By resolving structural heterogeneity, this in-depth analysis substantially improves the quality of the electron density maps, and provides a clear visualization of the isolated intermediates from I to M. The earliest photoproducts revealed by the deconvoluted maps suggest that a proton transfer uphill against 15 pH units is accomplished by the same physics governing the tablecloth trick. While the Schiff base is displaced at the beginning of the photoisomerization within ~30 fs, the proton stays due to its inertia. This affinity-independent early deprotonation builds up a steep proton concentration gradient that subsequently drives the directional proton conductance toward the extracellular medium. This mechanism fundamentally deviates from the widely adopted notion on multiple steps of chemical equilibrium driven by light-induced changes of proton affinity. The method of a numerical resolution of concurrent events from mixed observations is also generally applicable. Significance Statement Microorganisms can exploit solar energy to offset their cellular acidity from the environment by pumping protons outward under light illumination. The ability to transport ions across the cell membrane in response to light makes this family of small transmembrane proteins a highly desirable toolkit in development of new biotechnologies. It is important to understand how these ion pumps operate at the molecular level. This study finds that the outward proton conductance through bacteriorhodopsin, the most studied model system in the class, is driven by a steep concentration gradient of protons established in the light induced process rather than by an affinity gradient previously sought for decades. Summary of Revision This is the companion manuscript of another paper already published in PNAS Nexus (Ren, Photoinduced isomerization sampling of retinal in bacteriorhodopsin, PNAS Nexus, 1(3), 2022, 10.1093/pnasnexus/pgac103). The original version of this manuscript was submitted to PNAS Nexus on February 18, 2022. The manuscript was reviewed by three reviewers and the Decision Notification was received on April 5, 2022. I appealed the decision to reject the manuscript on May 28, 2022, and the appeal was accepted. A revised version of the manuscript was submitted on July 25, 2022, with an extensive response to the peer review. The editor sent the revised version and the response to peer review back to the three reviewers. Reviewer 3 declined to review the revised manuscript. The editor extended the invitation to several other scientists to review the revised manuscript. All of them declined to review. The second Decision Notification based on the opinions of Reviewers 1 and 2 was received on September 14, 2022. The revised manuscript, the supplementary materials, and all review documents are listed below in the Table of contents. Second revision is underway.