title: His75-Asp97 cluster in green proteorhodopsin.
abstract: The proteorhodopsin (PR) family found in bacteria near the ocean's surface consists of hundreds of PR variants color-tuned to their environment. PR contains a highly conserved single histidine at position 75, which is not found in most other retinal proteins. Using (13)C and (15)N MAS NMR, we were able to prove for green PR that His75 forms a pH-dependent H-bond with the primary proton acceptor Asp97, which explains its unusually high pK(a). The functional role of His75 has been studied using site-directed mutagenesis and time-resolved optical spectroscopy: Ultrafast vis-pump/vis-probe experiments on PR(H75N) showed that the primary reaction dynamics is retained, while flash photolysis experiments revealed an accelerated photocycle. Our data show the formation of a pH-dependent His-Asp cluster which might be typical for eubacterial retinal proteins. Despite its stabilizing function, His75 was found to slow the photocycle in wild-type PR. This means that PR was not optimized by evolution for fast proton transfer, which raises questions about its true function in vivo.
authors.affiliation: Institute of Biophysical Chemistry & Centre for Biomolecular Magnetic Resonance, Goethe-University Frankfurt, Frankfurt, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Hempelmann
authors.forename: Soraya
authors.initials: S
authors.lastname: Hölper
authors.forename: Mirka-Kristin
authors.initials: MK
authors.lastname: Verhoefen
authors.forename: Andreas C
authors.initials: AC
authors.lastname: Woerner
authors.forename: Thomas
authors.initials: T
authors.lastname: Köhler
authors.forename: Sarah-Anna
authors.initials: SA
authors.lastname: Fiedler
authors.forename: Nicole
authors.initials: N
authors.lastname: Pfleger
authors.forename: Josef
authors.initials: J
authors.lastname: Wachtveitl
authors.forename: Clemens
authors.initials: C
authors.lastname: Glaubitz
mesh_terms: D001224:Aspartic Acid / Q000737:chemistry*; D003116:Color; D006639:Histidine / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000592:standards; D008958:Models, Molecular; D012015:Reference Standards; D012243:Rhodopsin / Q000737:chemistry*; D025604:Rhodopsins, Microbial
chemical_list: D025604:Rhodopsins, Microbial; C413548:proteorhodopsin; D001224:Aspartic Acid; D006639:Histidine; D012243:Rhodopsin