Replacement of the methionine axial ligand in cytochrome c₅₅₀ by a lysine: effects on the haem electronic structure

Abstract

The prosthetic group of low-spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis-His and His^Met cytochromes to His^Lys co-ordination in the M100K mutant of Paracoccus versutus cytochrome c 550 . The electronic structure of the His^Lys haem is shown to be similar to that produced by His^cyanide co-ordination, such that NMR can be used to determine the geometry of the His ligand. ß

The Effects of Ligand Exchange and Mobility on the Peroxidase Activity of a Bacterial Cytochrome c upon Unfolding

“…The latter may indicate that the histidinehistidine form giving rise to the broad signals assigned as B 1 and B 2 is in exchange with multiple unfolded species, whereas this is no longer the case in the presence of CN À . Two peaks effect on the electronic structure of the heme in terms of heme methyl chemical shifts, [54] this provides further evidence that A 1 and A 2 arise from lysine coordination.…”

“…Upon addition of KCN (final concentration 3 mm) to the lysine-modified protein in the presence of Gdm·HCl, peaks B 1 and B 2 decrease dramatically in intensity and heme methyl signals of a CN À -bound form predominate ( Figure 7). [54] It is noted that peaks B 1 and B 2 are still present but are of much lower intensity and appear to be sharper ( Figure 7). The latter may indicate that the histidinehistidine form giving rise to the broad signals assigned as B 1 and B 2 is in exchange with multiple unfolded species, whereas this is no longer the case in the presence of CN À .…”

Sulfur and Selenium: The Role of Oxidation State in Protein Structure and Function

“…[89][90][91] It is also found as a metal ligand in many proteins such as ascorbate oxidase (copper) [92] and various cytochromes (iron). [93] As an example for oxidized methionine, the bacterial catalase from Proteus mirablis PR (a peroxide-resistant 56-kDa enzyme) contains a heme group found to interact with a methionine sulfone, which could prevent access of large substrates or inhibitors to the ironcontaining active site. [94] Metal binding to cysteine leads us to another group of redox enzymes that contain redox-inactive cysteine residues at the active site.…”

Recent developments in the 13 C NMR spectroscopic analysis of paramagnetic hemes and heme proteins

“…This experiment has found widespread use in the identification of 1 H and 13 C resonances originating from protonated carbon atoms in paramagnetic heme centers [54,55,56]. However, resonances not resolved from the paramagnetic envelope are often difficult to assign, even with the aid of the HMQC experiment [57,58] Biosynthetic preparation of 13 C-labeled heme A biosynthetic strategy that takes advantage of developments in recombinant DNA methodology and knowledge of the heme biosynthesis pathway has been developed to prepare isotopically enriched heme [59]. The first committed precursor in the biosynthetic pathway of heme is δ-aminolevulinic acid (ALA) [60,61].…”