High-resolution structural studies of the D85S mutant of bacteriorhodopsin, bR(D85S), are
of special interest because this mutant is likely to adopt a constitutively 0-like protein conformation
in the anion-free ground-state. The key features that bR(D85S) and the 0-intermediate have in
common are the protonated state of the Schiff base and the fact that residue 85 is uncharged and yet
offers an hydroxyl group that is available for "solvating" a bound anion. The "acid blue" form of bR,
in which D85 is protonated due to the low pH [5,6], may also be in a constitutively O-like state, for
the same reasons. It is unlikely, however, that well-diffracting crystals of wt bR could be grown at a
pH value of 2 or lower, which is needed to populate the protonated, blue state of wt bR at high
occupancy.
Both bR(D85S) and the acid-blue wt protein bind chloride ions and pump them across the
cell membrane when illuminated [5,6]. These observations, along with the fact that halorhodopsin - a
related membrane protein - serves physiologically as a chloride-ion pump, make it natural to
consider that wt bR may also be an anion pump, but one that has high specificity for hydroxyl ions.
High-resolution crystal structures of bR(D85S) have been published recently for which the
protein is in either the anion-free (blue) state [7,8] or in a purple state with either bromide ion [9] or
nitrate ion [8] in the internal binding pocket. The analysis of these structures has so far focused,
however, on describing (1) the favorable interactions that this binding pocket offers to either water
molecules or to small anions [8], (2) the conformational differences that are observed on the
extracellular side of the protein when the O-like, anion-free protein is compared to the resting state,
bR568 structure [7], (3) the fact that the extracellular side of the protein "returns" to a bR568-like
conformation when anions are bound adjacent to the protonated Schiff base [9], and (4) the way in
which 13-cis photoisomerization is likely to raise the electrochemical potential of a bound anion [9].
In addition to reviewing again the previously described character of the anion-binding pocket in
bR(D85S) in comparison to the corresponding site in wt bR, we now consider the role that the side

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