Document Detail


The role of back-reactions and proton uptake during the N----O transition in bacteriorhodopsin's photocycle: a kinetic resonance Raman study.
MedLine Citation:
PMID:  2169875     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The kinetics of bacteriorhodopsin's photocycle have been analyzed at pH 5, 6, 7, 8, and 8.6 by using time-resolved resonance Raman spectroscopy. The concentrations of the various intermediates as a function of time were determined by following their resonance Raman intensities using 502-nm (L550, N550, BR568), 458-nm (M412), and 752-nm (O640) excitation. The spectral contributions to the pump + probe data from each intermediate were quantitatively separated by least-squares decomposition. These relative concentrations were then converted to absolute concentrations by using a conservation of molecules constraint. This enabled the unambiguous refinement of a variety of kinetic models to find the simplest one that accurately describes the data. The kinetic data, including the biphasic decay of L550 and M412, are best reproduced by a sequential scheme including back-reactions (BR----L----M----N----O----BR). In addition, the kinetics of the L----M and N----O steps are found to be pH-dependent. Both the forward and reverse rate constants connecting L550 and M412 increase with pH, confirming earlier proposals of catalyzed Schiff base deprotonation at alkaline pH. Below pH 7, the N550----O640 rate constant is independent of pH, but it decreases linearly with pH above 7. This indicates that the protein must pick up a proton during the N550----O640 transition and that this process becomes rate determining above pH 7. There must, therefore, be an intermediate between N550 and O640 which we denote as N+550. A molecular graphics model is presented which incorporates these observations into a mechanism for proton pumping.
Authors:
J B Ames; R A Mathies
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Publication Detail:
Type:  Comparative Study; Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Biochemistry     Volume:  29     ISSN:  0006-2960     ISO Abbreviation:  Biochemistry     Publication Date:  1990 Aug 
Date Detail:
Created Date:  1990-11-20     Completed Date:  1990-11-20     Revised Date:  2007-11-14    
Medline Journal Info:
Nlm Unique ID:  0370623     Medline TA:  Biochemistry     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  7181-90     Citation Subset:  IM    
Affiliation:
Department of Chemistry, University of California, Berkeley 94720.
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MeSH Terms
Descriptor/Qualifier:
Bacteriorhodopsins / chemistry,  metabolism*,  radiation effects
Halobacterium / analysis
Hydrogen-Ion Concentration
Kinetics
Photochemistry
Protein Conformation
Protons
Spectrum Analysis, Raman
Grant Support
ID/Acronym/Agency:
GM 27057/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Protons; 53026-44-1/Bacteriorhodopsins

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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