PH-sensitive vibrational probe reveals a cytoplasmic protonated cluster in bacteriorhodopsin

Victor A Lorenz-Fonfria,Mattia Saita,Tzvetana Lazarova,Ramona Schlesinger,Joachim Heberle

doi:10.1073/pnas.1707993114

Abstract

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium. Here, by selectively monitoring vibrational changes of buffer molecules with a temporal resolution of 6 µs, we have traced proton release and uptake events in the light-driven proton-pump bacteriorhodopsin and correlate these to other molecular processes within the protein. We demonstrate that two distinct chemical entities contribute to the temporal evolution and spectral shape of the continuum band, an unusually broad band extending from 2,300 to well below 1,700 cm-1 The first contribution corresponds to deprotonation of the proton release complex (PRC), a complex in the extracellular domain of bacteriorhodopsin where an excess proton is shared by a cluster of internal water molecules and/or ionic E194/E204 carboxylic groups. We assign the second component of the continuum band to the proton uptake complex, a cluster with an excess proton reminiscent to the PRC but located in the cytoplasmic domain and possibly stabilized by D38. Our findings refine the current interpretation of the continuum band and call for a reevaluation of the last proton transfer steps in bacteriorhodopsin.

Highlights

Infrared spectroscopy has been used in the past to probe the dynamics of internal proton transfer reactions taking place during the functional mechanism of proteins but has remained mostly silent to protonation changes in the aqueous medium
Gerwert and coworkers [38, 39] showed that the proton release complex (PRC), the elusive group releasing a proton to the EC medium in BR (Fig. 1B), was characterized by an unusually broad band extending from ∼2,300 cm−1 to well below 1,700 cm−1 (Fig. 1C), known as the continuum band
How to conceive the formation of the N2 intermediate with τ ≈ 1.5 ms with a reprotonated D96, when proton uptake from the medium occurs with τ ≈ 4.5 ms? if the continuum band with τ ≈ 1.5 ms reports on the deprotonation of a protonated local area network (LAN), where is the corresponding proton transferred to? To answer these two questions we propose that ionic D96 gets a proton in the N1-to-N2 transition from the proton uptake complex (PUC), not from the CP medium as currently accepted

Summary

BIOPHYSICS AND COMPUTATIONAL BIOLOGY

A relevant example to test the potential of buffer molecules to probe the dynamics of proton release and uptake events is the light-driven proton-pump bacteriorhodopsin (BR) [27]. This well-known transmembrane protein powers halophilic archaebacteria under low oxygen tension [28]. Gerwert and coworkers [38, 39] showed that the proton release complex (PRC), the elusive group releasing a proton to the EC medium in BR (Fig. 1B), was characterized by an unusually broad band extending from ∼2,300 cm−1 to well below 1,700 cm−1 (Fig. 1C), known as the continuum band

BR K

Results and Discussion

General Discussion and Conclusions