Ligation and Reactivity of Methionine-Oxidized Cytochrome c

Abstract

Met80, one of the heme iron ligands in cytochrome c (cyt c) is readily oxidized to Met sulfoxide (Met-SO) by several biologically-relevant oxidants. The modification has been suggested to impact both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO implying the redox-linked ligand switch in the modified protein. Binding studies with a model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K#) and Met-SO is more than three orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)22+. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for non-switchable M80A and M80K#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (pKa=6.3±0.1) increasing intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells.

Lysine carbonylation is a previously unrecognized contributor to peroxidase activation of cytochrome c by chloramine-T

“…Those early studies17,48,50,51 cemented the widely held belief that Met80 oxidation, without any other covalent changes, generates an active peroxidase 17,18,37,45–47. As a result, CT-cyt c has become a widely used model for peroxidase-activated cyt c 17,37,38,47…”

“…The most straightforward method to rupture the Met80–Fe bond is via Met oxidation to methionine sulfoxide (MetO). It is widely believed that this oxidative modification is sufficient for generating the open distal site that is required for peroxidase activation of cyt c in vitro and in vivo 17,18,37,38,44–47. In other words, this Met80-centric view envisions a simple causal relationship between MetO formation and peroxidase activity 17,18,37,38,45–47…”

“…Several lines of evidence hint at the involvement of additional factors. (1) Disruption of the Met80–Fe bond tends to produce “alkaline” ligation scenarios, where even at near-neutral pH the distal site becomes occupied by side chains such as Lys72/73 19,35,36,38. These alternative ligation scenarios are promoted by the flexible nature of the 71–85 Ω loop (Fig.…”

Wheel and Deal in the Mitochondrial Inner Membranes: The Tale of Cytochromecand Cardiolipin

“…While tyrosine residues can covalently cross-link or undergo oxidation to dihydroxyphenylalanine (DOPA) and subsequently to quinones, lysine residues can undergo carbonylation [152,168,169]. These changes occur when H 2 O 2 is added to Cc samples [80,152,[170][171][172][173].…”

Discovery of a functional, contracted heme-binding motif within a multiheme cytochrome

“…UV/Vis electronic absorption spectrum of the as-isolated (fully oxidized) KsTH displayed a broad Soret band with a maximum at 409 nm while, upon reduction, the Soret band maximum shifted to 418 nm with a pronounced increase in amplitude and decrease in half width. Absence of a shoulder at 437 nm and 564 nm (Figure 4A)ruled out the possibility of a hydroxide ligand for any of the hemes of KsTH(31) and absence of a charge transfer band at 695 nm most likely excluded methionine as a heme axial ligand(27). Upon reduction, the Soret bands of His/Lys, His/His, and His/Cys ligated hemes shift from 408 nm to 417 nm(32), 407 nm to 420 nm(30), and 418 nm to 416 nm(33),respectively.…”