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What is more, the indicator has two proton-binding sites: the oxyanion of the xanthene ring and the carboxylate of the benzene as independent sites, which can also exchange protons among themselves.
Such a low pH value suggests that at higher pH the protonation of a carboxylate , forming a hydrogen bond, would break this interaction leading to a destabilization and a change in the heme pocket crucial to catalysis.
Besides requiring vitamin K, these extrinsic factors need carbon dioxide and oxygen for carboxylation .
OAA would be produced by carboxylation of phosphoenol pyruvate in a reaction catalysed by guard cell PEP carboxylase.
Lack of proton-pumping activity has also been found in heterologously expressed Neurospora rhodopsin, which has carboxylated residues at both the Schiff base proton donor and acceptor positions, as does GtR1.
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