Photochemical reaction cycle and proton transfers in Neurospora rhodopsin.
It was recently found that NOP-1, a membrane protein of Neurospora crassa, shows homology to haloarchaeal rhodopsins and binds retinal after heterologous expression in Pichia pastoris. We report on spectroscopic properties of the Neurospora rhodopsin (NR). The photocycle was studied with flash photolysis and time-resolved Fourier-transform infrared spectroscopy in the pH range 5-8. Proton release and uptake during the photocycle were monitored with the pH-sensitive dye, pyranine. Kinetic and spectral analysis revealed six distinct states in the NR photocycle, and we describe their spectral properties and pH-dependent kinetics in the visible and infrared ranges. The phenotypes of the mutant NR proteins, D131E and E142Q, in which the homologues of the key carboxylic acids of the light-driven proton pump bacteriorhodopsin, Asp-85 and Asp-96, were replaced, show that Glu-142 is not involved in reprotonation of the Schiff base but Asp-131 may be. This implies that, if the NR photocycle is associated with proton transport, it has a low efficiency, similar to that of haloarchaeal sensory rhodopsin II. Fourier-transform Raman spectroscopy revealed unexpected differences between NR and bacteriorhodopsin in the configuration of the retinal chromophore, which may contribute to the less effective reprotonation switch of NR.
Brown
L S
LS
Department of Physiology & Biophysics, University of California, Irvine, California 92697, USA. lsbrown@uci.edu
Dioumaev
A K
AK
Lanyi
J K
JK
Spudich
E N
EN
Spudich
J L
JL
D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins; D019617:Halobacterium salinarum / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008027:Light; D016297:Mutagenesis, Site-Directed; D009492:Neurospora crassa / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D010641:Phenotype; D010777:Photochemistry; D010782:Photolysis; D010843:Pichia / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D013053:Spectrophotometry; D017550:Spectroscopy, Fourier Transform Infrared
D002352:Carrier Proteins; D005656:Fungal Proteins; C401582:NOP-1 protein, Neurospora crassa; D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D012243:Rhodopsin