Evidence that a formyl-substituted iron porphyrin is the prosthetic group of myeloperoxidase: magnetic circular dichroism similarity of the peroxidase to Spirographis heme-reconstituted myoglobin.

Abstract

To probe the identity of the active site hemetype prooshetic group ofmyeloperoxidase, whose (5,6,9,10), the spectroscopic properties of analogous derivatives with the same oxidation and ligation states can be used to probe the similarity or difference in their prosthetic group structures. MCD spectral comparison between myoglobin reconstituted with Spirographis heme (Spirographis Mb) and MPO in this study reveals considerable spectral similarities between parallel derivatives of these two proteins, especially in the ferric high-spin native form and the ferrous low-spin complexes. Given the repeatedly demonstrated ability of MCD spectroscopy to identify or distinguish two apparently similar chromophores more definitively than electronic absorption spectroscopy (18-20), the present finding provides strong support for a formyl-substituted porphyrin as the structure of the prosthetic group macrocycle of MPO. EXPERIMENTAL PROCEDURES Spirographis Mb was prepared from horse heart apo-Mb and Spirographis heme as described by Sono and Asakura (16,21,22). MPO was purified from bovine spleen as described (23). Metal-free trans-octaethylchlorin (GEC) was obtained by a published method (24) and metallated to produce Fe-(OEC)Cl using a modification (25) of the direct iron insertion method (26). All chemicals were reagent grade and were obtained from Aldrich or Sigma. N-Methylimidazole and benzene were further purified by distillation.The reduced pyridine hemochromogen [the ferrous bis(pyridine) adduct] of Spirographis heme (and Mb) or MPO was generated by dissolving the heme or diluting the concentrated protein stock solution into a pyridine/NaOH solution [25-35% pyridine (vol/vol) and 0

Spectral Analysis of Lactoperoxidase

“…Note that its pattern of bands is reversed relative to those of LPO and Mb, with the most intense positive band at low energy and the most intense negative band at high energy. As previously discussed (11,12), this reversed MCD pattern is the expected one for an iron chlorin or a formyl-substituted porphyrin. Given that neither of these is consistent with the new reports of the heme m structure of MPO (see Fig.…”

Interactions between Heme d and Heme b595 in Quinol Oxidase bd from Escherichia coli: A Photoselection Study Using Femtosecond Spectroscopy

“…Figure 5 in ref 22). We note that a split Soret band was also observed in a CO-ligated octo-ethyl chlorin and other six-coordinated low-spin ferrous chlorines (37).…”

The Sulfonium Ion Linkage in Myeloperoxidase

“…In the past spectroscopic and chemical evidence for the presence of a formyl-containing heme in MPO has been presented (7,8,49,50). In terms of the present knowledge this can now be explained in the following way.…”