title: Breaking the carboxyl rule: lysine 96 facilitates reprotonation of the Schiff base in the photocycle of a retinal protein from Exiguobacterium sibiricum.
abstract: A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.
authors.affiliation: From the Department of Physiology and Biophysics, University of California, Irvine, California 92697,. Electronic address: balashov@uci.edu.
authors.forename: Sergei P
authors.initials: SP
authors.lastname: Balashov
authors.affiliation: the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia, and. Electronic address: lpetr65@yahoo.com.
authors.forename: Lada E
authors.initials: LE
authors.lastname: Petrovskaya
authors.affiliation: From the Department of Physiology and Biophysics, University of California, Irvine, California 92697.
authors.forename: Eleonora S
authors.initials: ES
authors.lastname: Imasheva
authors.affiliation: the Department of Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: Evgeniy P
authors.initials: EP
authors.lastname: Lukashev
authors.affiliation: From the Department of Physiology and Biophysics, University of California, Irvine, California 92697.
authors.forename: Andrei K
authors.initials: AK
authors.lastname: Dioumaev
authors.affiliation: From the Department of Physiology and Biophysics, University of California, Irvine, California 92697.
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Wang
authors.affiliation: the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia, and.
authors.forename: Sergey V
authors.initials: SV
authors.lastname: Sychev
authors.affiliation: the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia, and; the Department of Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: Dmitriy A
authors.initials: DA
authors.lastname: Dolgikh
authors.affiliation: the Department of Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: Andrei B
authors.initials: AB
authors.lastname: Rubin
authors.affiliation: the Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Moscow 117997, Russia, and; the Department of Biology, Lomonosov Moscow State University, Moscow 119991, Russia.
authors.forename: Mikhail P
authors.initials: MP
authors.lastname: Kirpichnikov
authors.affiliation: From the Department of Physiology and Biophysics, University of California, Irvine, California 92697,. Electronic address: jklanyi@uci.edu.
authors.forename: Janos K
authors.initials: JK
authors.lastname: Lanyi
mesh_terms: D000042:Absorption / Q000528:radiation effects; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000528:radiation effects; D017666:Deuterium Oxide / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006217:Halobacterium / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects / Q000528:radiation effects; D007700:Kinetics; D008027:Light; D008081:Liposomes / Q000378:metabolism; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D011522:Protons; D012545:Schiff Bases / Q000378:metabolism*; D019810:Sodium Azide / Q000494:pharmacology; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D050505:Mutant Proteins; D011522:Protons; D012545:Schiff Bases; D019810:Sodium Azide; D017666:Deuterium Oxide; D008239:Lysine; D000409:Alanine
keywords: Bioenergetics; Internal Proton Donor; Photobiology; Proton Pumps; Proton Transport; Retinal Proteins; Spectroscopy