Fluorescence properties of tryptophan residues in the monomeric d-chain of Glossoscolex paulistus hemoglobin: an interpretation based on a comparative molecular model

Abstract

The primary structure of the 142 residue Glossoscolex paulistus d-chain hemoglobin has been determined from Edman degradation data of 11 endo-Glu-C peptides and 11 endo-Lys-C peptides, plus the results of Edman degradation of the intact globin. Tryptophan occupies positions 15, 33 and 129. Homology modeling allowed us to assign the positions of these Trp residues relative to the heme and its environment. The reference coordinates of the indole rings (average coordinates of the C ε2 and C δ2 atoms) for W15 and W129 were 16.8 and 18.5 Å, respectively, from the geometric center of the heme, and W33 was located in close proximity to the heme group at a distance which was approximately half of that for W15 and W129. It was possible to identify three rotamers of W33 on the basis of electrostatic and Van der Waals energy criteria. The calculated distances from the center of the heme were 8.3, 8.4 and 9.1 Å for Rot1, Rot2 and Rot3, respectively. Radiationless energy transfer from the excited indole to the heme was calculated on the basis of Förster theory. For W33, the distance was more important than the orientation factor, κ 2, due to its proximity to the heme. However, based on κ 2, Rot2 (κ 2=0.945) was more favorable for the energy transfer than Rot1 (κ 2=0.433) or Rot3 (κ 2=0.125). In contrast, despite its greater distance from the heme, the κ 2 of W129 (2.903) established it as a candidate to be more efficiently quenched by the heme than W15 (κ 2=0.191). Although the Förster approach is powerful for the evaluation of the relative efficiency of quenching, it can only explain pico- and sub-nanosecond lifetimes. With the average lifetime, 〈τ〉=3 ns, measured for the apomonomer as the reference, the lifetimes calculated for each emitter were: W33-1 (1 ps), W33-2 (2 ps), W33-3 (18 ps), W129 (100 ps), and W15 (600 ps). Experimentally, there are four components for oxymonomers at pH 7: two long ones of 4.6 and 2.1 ns, which contribute approximately 90% of the total fluorescence, one of 300 ps (4%), and the last one of 33 ps (7.4%). It is clear that the equilibrium structure resulting from homology modeling explains the sub-nanosecond fluorescence lifetimes, while the nanosecond range lifetimes require more information about the protein in solution, since there is a significant contribution of lifetimes that resemble the apo molecule.