Studies on the binding of nitrogenous bases to protoporphyrin IX iron(II) in aqueous solution at high pH values

Abstract

Studies are reported on the formation of low-spin six-coordinate [Fe(PPIX)L₂] complexes from iron(II) protoporphyrin where L is one of a series of nitrogenous ligands (aliphatic, aromatic or heterocyclic). The bonding constants have been determined by titration of the metal complex with these ligands and are compared in relation to previous studies. The adduct formation was monitored utilising optical spectroscopy. In addition, Mӧssbauer spectroscopic experiments were conducted to monitor the electronic environment around the central iron atom in these complexes. The two complementary spectroscopic methods indicated that all nitrogen ligands formed low-spin octahedral complexes. The magnitude of the overall binding constants (β₂ values) are discussed and related to (a) the pK_a values of the free ligands and (b) the Mössbauer parameter ΔE_Q, which represents the quadrupole splitting of the haem iron. The β₂ and ΔE_Q values are also discussed in terms of the structure of the ligand. Cooperative binding was observed for nearly all the ligands with Hill coefficients close to 2 for iron(II) protoporphyrin; one of these ligands displayed a much greater affinity than any we previously studied, and this was a direct consequence of the structure of the ligand. Overall conclusions on these and previous studies are drawn in terms of aliphatic ligands versus aromatic ring structures and the absence or presence of sterically hindered nitrogen atoms. The implications of the work for the greater understanding of haem proteins in general and in particular how the nitrogenous ligand binding results are relevant to and aid the understanding of the binding of inhibitor molecules to the cytochrome P₄₅₀ mono-oxygenases (for therapeutic purposes) are also discussed. Changes in the electronic absorption spectra of five-coordinate [Fe(II)(PPIX)(2-MeIm)] that occurred as the temperature was lowered from room temperature to 78° K.

Keywords: Hill coefficients; Mossbauer spectroscopy; Nitrogenous ligands; Protoporphyrin IX iron(II); Stability constants.

Fig. 1

The electronic absorption spectra of [Fe(II)(PPIX)] in the presence of a large excess of 2-MeIm in 50:50v water/-glycerol. A Five coordinate [Fe(II)(PPIX)(2-MeIm)] at room temperature; B Changes in the electronic absorption spectra that occurred as the temperature was lowered to 78° K; C Six coordinate [Fe(II)(PPIX)(2-MeIm₂)] at78° K

Fig. 2

A plot of log β₂ values against the pK_a values of the free ligands. The pyridines (6-ring coloured blue) can be fitted to a trend line, the imidazole and 5-membered rings (coloured red) would straddle this trend line, whereas the other six-membered rings (green triangles) are widely spread across the trend. The aliphatic ligands shown in black are well separated and in a tight group

Fig. 3

A plot of log β₂ against quadrupole splitting (ΔE_Q) for the Fe(II)(PPIX)L₂ complexes. The six-membered rings mainly have the largest ΔE_Q values. The pyridine rings are high and to the right. The five-membered rings are predominantly on the left they have smaller ΔE_Q values and their shape and bonding properties allows them to get close to the Fe(II) atom. The aliphatic ligands are all at the bottom