STRUCTURE OF DEOXYHEMOGLOBIN OF THE ANTARCTIC FISH PAGOTHENIA-BERNACCHII WITH AN ANALYSIS OF THE STRUCTURAL BASIS OF THE ROOT EFFECT BY COMPARISON OF THE LIGANDED AND UNLIGANDED HEMOGLOBIN STRUCTURES

We have determined the structure of deoxyhaemoglobin from the antarcti c fish Pagothenia bernacchii at pH 6.2 to a resolution of 2.2 Angstrom with X-ray data from a twinned crystal deconvoluted so as to approxim ate data from a single crystal. The R-factor between the (twinned) mod el and the observed data is 16% for reflections used in refinement and 22% for reflections not used in refinement. The T (deoxy) structure w as compared with the R (liganded) structure at pH 8.0 in an attempt to understand the structural basis of the greater affinity for hydrogen ions of T,relative to R, that comprises the Root effect. Up to half of the effect can be attributed to interaction of the residues Asp95 (G1 )alpha and Asp101 (G3)beta: in R the residues are far apart and their carboxyl groups are unprotonated, but the shift at the alpha(1) beta(2 ) interface that accompanies the R to T transition brings them so clos e that they appear to share a proton between them. The proximity of As p99 (G1)beta may contribute to the required raising of the pK(a) value s of the other two Asp residues. These and neighbouring residues are s ufficiently conserved in the haemoglobins of trout (component IV), car p and bluefin tuna, all of which exhibit the Root effect, for the same mechanism to apply. However, the environment is equally conserved in haemoglobins of Trematomus newnesi (major component) and trout (compon ent I), which do not exhibit the Root effect, so that the structural f actors controlling the Asp-Asp interaction remain unclear. No other re sidue appears to undergo an R to T change in the immediate neighbourho ods that could account for any significant portion of the Root effect, so at least half of the effect must result either from long-range ele ctrostatic interactions or from a large number of local interactions. (C) 1995 Academic Press Limited