Energetics of a hydrogen bond (charged and neutral) and of a cation-pi interaction in apoflavodoxin

Anabaena apoflavodioxin contains a single histidine residue (H34) that inte racts with two aromatic residues (F7 and Y47). The histidine and phenylalan ine rings are almost coplanar and they can establish a cation-pi interactio n when the histidine is protonated. The histidine and tyrosine side-chains are engaged in a hydrogen bond, which is their only contact. We analyse the energetics of these interactions using pK(a)-shift analysis, double-mutant cycle analysis at two pH values, and X-ray crystallography. The H/F intera ction is very weak when the histidine is neutral, but it is strengthened by 0.5 kcal mol(-1) on histidine protonation. Supporting this fact, the histi dine pK(a) in a FR mutant is 0.4 pH units lower than in wild-type. The stre ngth of the H/Y hydrogen bond is 0.7 kcal mol(-1) when the histidine is cha rged, and it becomes stronger (1.3 kcal mol(-1)) when the histidine is neut ral. This is consistent with our observation that the (H34)N-epsilon 2-OH(Y 47) distance ij slightly shorter in the apoflavodoxin structure at FH 9.0 t han in the previously reported structure at pH 6.0. It is also consistent w ith a histidine pK(a) value 0.6 pH units higher in a Y47F mutant than in th e wild-type protein. We suggest that the higher stability of the neutral hy drogen bond could be due to a higher desolvation penalty of the charged hyd rogen bond that would offset its more favourable enthalpy of formation. The relationship between hydrogen bond strength and the contribution of hydrog en bonds to protein stability is discussed. (C) 1999 Academic Press.