Steady-state kinetics studies on (A) heme destruction and (B) peroxidase activity vs pH change of wild-type cyt <i>c</i> and its Y67H mutant.

<p>Steady-state kinetics studies on (A) heme destruction and (B) peroxidase activity vs pH change of wild-type cyt <i>c</i> and its Y67H mutant.</p

Structure of the heme region of cyt <i>c</i> and the location of Tyr67 in the structure (pdb code: 1YIC), showing the hydrogen bond network formed by amino acids His18, Tyr67, Met80, Asn52 and Thr78.

<p>Structure of the heme region of cyt <i>c</i> and the location of Tyr67 in the structure (pdb code: 1YIC), showing the hydrogen bond network formed by amino acids His18, Tyr67, Met80, Asn52 and Thr78.</p

Experimental restraints and structural statistics for cyt <i>c</i> Y67H variant.

a<p>The programs PROCHECK and PROCHECK-NMR were used to check the overall quality of the structure and GLY and Pro are excluded from the Ramachandran analysis.</p>b<p>For the PROCHECK statistic, less than 10 bad contacts per 100 residues, and an overall G-factor larger than −0.5 are expected for a good quality structure.</p><p>Experimental restraints and structural statistics for cyt <i>c</i> Y67H variant.</p

1D ¹H-NMR spectra of cyt <i>c</i>.

<p>(A) The high-field shifted region of the native cyt c (down) and its Y67H variant (upper) spectra. (B) The down-field shifted region of the native cyt <i>c</i> (down) and its Y67H variant (upper) spectra. The peaks in 1D ¹H-NMR spectrum were assigned as (1a) His18 Hε1, (2a) His71 Hε1, (3a) His71 Hδ2, (4a) heme 8-CH₃, (5a) His18 Hδ2, (6a) heme 3-CH₃, (7a) heme 5-CH₃, (8a) His18 Hβ2, (9a) heme 1-CH₃ and (10a) heme 7-Hα2, respectively. The peaks in 1D ¹H-NMR spectrum of native cyt <i>c</i> were assigned as (1b) His18 Hε1, (2b) Met80 ε-CH₃, (3b) Met80 Hγ, (4b) heme 8-CH₃, (5b) heme 3-CH₃, (6b) His18 Hδ2, (7b) heme 7-Hα2, (8b) His18 Hβ2, (9b) heme 7-Hα1 and (10b) His18 Hδ1, respectively.</p

Structural Basis for Cytochrome <i>c</i> Y67H Mutant to Function as a Peroxidase

<div><p>The catalytic activity of cytochrome <i>c</i> (cyt <i>c</i>) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt <i>c</i> Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe³⁺ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H₂O₂ by functioning as an acidic catalyst.</p></div

New assignments of Y67H mutant.

<p>New assignments of Y67H mutant.</p

Heme destruction of the wild-type cyt <i>c</i> (A) and its Y67H variant (B) as indicated by dissipation of the Soret band.

<p>Spectra were scanned every 20 sec, and the arrows indicate the direction of change. The concentrations of H₂O₂ were 10 mM in (A) and 1 mM in (B), the pH condition in both cases is 6.0.</p

Upon overlaying Cα atoms in second structural region and heme backbone atoms, the conformational comparison: (A) between wild-type cyt <i>c</i> (grey, pdb code 1YIC) and its Y67H variant (green); (B) between the Y67F (<i>magenta</i>, pdb code 1CTY) and Y67H (green) mutants; (C) between Y67H (green) and alkaline form (red, pdb code 1LMS).

<p>Upon overlaying Cα atoms in second structural region and heme backbone atoms, the conformational comparison: (A) between wild-type cyt <i>c</i> (grey, pdb code 1YIC) and its Y67H variant (green); (B) between the Y67F (<i>magenta</i>, pdb code 1CTY) and Y67H (green) mutants; (C) between Y67H (green) and alkaline form (red, pdb code 1LMS).</p

The possible mechanism for guaiacol reaction with H₂O₂ catalyzed by the cyt <i>c</i> Y67H mutant at pH 4.0.

<p>The possible mechanism for guaiacol reaction with H₂O₂ catalyzed by the cyt <i>c</i> Y67H mutant at pH 4.0.</p

The position comparison of some key residues (including Asn52, Trp59, Tyr67, Lys73, Thr78, Phe82) relative to heme ring upon overlaying heme ring: (A) between the native cyt <i>c</i> (<i>grey</i>, pdb code 1YIC) and its Y67H variant (<i>green</i>); (B) between the cyt <i>c</i> Y67H variant (<i>green</i>) and the Y67F variant (<i>magenta</i>, pdb code 1CTY); (C) between cyt <i>c</i> Y67H variant (<i>green</i>) and the alkaline form of cyt <i>c</i> (<i>red</i>, pdb code 1LMS).

<p>The position comparison of some key residues (including Asn52, Trp59, Tyr67, Lys73, Thr78, Phe82) relative to heme ring upon overlaying heme ring: (A) between the native cyt <i>c</i> (<i>grey</i>, pdb code 1YIC) and its Y67H variant (<i>green</i>); (B) between the cyt <i>c</i> Y67H variant (<i>green</i>) and the Y67F variant (<i>magenta</i>, pdb code 1CTY); (C) between cyt <i>c</i> Y67H variant (<i>green</i>) and the alkaline form of cyt <i>c</i> (<i>red</i>, pdb code 1LMS).</p