PMC:103662 / 21346-25528
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{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/103662","sourcedb":"PMC","sourceid":"103662","source_url":"https://www.ncbi.nlm.nih.gov/pmc/103662","text":"Fast proton release in pR\nUnder the same conditions where M is observed (pH 9.5 and in 1% DHPC), pR undergoes fast proton release during its photocycle (Fig. 5). The pH indicator dye Cresol Red was used to detect pH changes in the bulk aqueous phase. These turn out to be similar to those observed for bR in the pH range 5.5–10. After photoexcitation, pR (like bR, presumably) ejects a H+ from a residue near its extracellular surface decreasing the pH of the solution. When the N → O transition takes place in bR, H+ is taken up from the medium, raising the pH once again. The H+ signals from pR measured with Cresol Red occur on a time scale similar to that assignable to M and N decay, returning to baseline about 1 s after photolysis.\nThere is a clear kinetic correlation between M (and/or N) intermediate formation and fast H+ release in pR. The linkage between these two phenomena is further supported by the observation that neither a transient 400-nm absorbance increase, nor fast H+ release, is shown to occur at pH 8.0 and below. Nor is either observed in the absence of a reconstituting lipid (DHPC in these experiments).\nIn bR, the ejected proton is thought to originate from a triad of amino acids, R82-E194-E204. However, in pR a homolog of only one of these three residues (the arginine) is present. This raises doubts about previous conclusions regarding the specific roles of these 3 residues in fast H+ proton release, in both pR and bR. In particular, the apparently obligatory roles of E204 and E194 in fast H+ release in bR are not matched in pR. Therefore, even in bR it is less likely that these groups themselves change protonation state between bR and M to provide the H+ released to the bulk medium. Instead, it now seems more likely that E204 and E194 merely help to lower the pKa of the H+ release group from above 8, the apparent value for pR, into the vicinity of 6 for bR. It also seems very unlikely that the specific structural configuration of 2 carboxylic acid groups and arginine in bR could be conserved in pR, even if, as suggested previously [1], other surface carboxylic acids in pR could substitute in some ways for the roles of E194 and E204 in bR.\nThe only way that the H+ release mechanism can be strongly conserved between bR and pR is if arginine itself serves as the principal donor group for fast H+ release in both, with nearby residues (such as E194 and E204) merely modulating the pKa of the arginine in the M intermediate. However, it remains unclear how the pKa of Arg-82 in bR could be made sufficiently low in its M intermediate to serve as the H+ release group at pH values down to 6.0.\nAlternatively, it is possible that the fast H+ release we observe from pR at pH 9.5 may differ from that in bR. One possibility is that in pR, the released proton could come directly from the chromophore counterion, Asp-97. This would be consistent with a proposed mechanism for fast H+ release that has been observed above pH 10 in the bR mutant E194Q [14]. In this mutant, Asp-85 was detected by low-temperature infrared difference spectroscopy to be deprotonated only in the N intermediate, and not in M [15]. It is not clear yet whether Asp-85 deprotonation in an N-like state could account for the proton-release kinetics of pR (Fig. 5), or whether the M intermediate itself might have a partially-deprotonated Asp-85.\nThe reason for the requirement of DHPC in M intermediate formation and fast proton release is unclear. Delipidated bR in octylglucoside is fully capable of M formation and presumably proton release, although with altered kinetics [12,16]. The requirement for pR to be in lipid to show fast H+ release and M formation stems either from a protein/lipid interaction needed to establish a stable, active tertiary structure, or from the need for the phosphate group in DHPC to act as a proton release group. The latter seems unlikely due to the DHPC molecule being zwitterionic at pH 9.5, with no proton on the trimethyl-modified nitrogen of the choline. Hence, the DHPC most likely interacts with the protein to effect minor structural changes needed to place the active site residues in their functional configuration.","divisions":[{"label":"title","span":{"begin":0,"end":25}},{"label":"p","span":{"begin":26,"end":738}},{"label":"p","span":{"begin":739,"end":1132}},{"label":"p","span":{"begin":1133,"end":2190}},{"label":"p","span":{"begin":2191,"end":2642}},{"label":"p","span":{"begin":2643,"end":3366}}],"tracks":[]}