Ligation and Reactivity of Methionine-Oxidized Cytochrome <i>c</i>

Met80, one of the heme iron ligands in cytochrome <i>c</i> (cyt <i>c</i>), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt <i>c</i> (Met-SO cyt <i>c</i>) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well as kinetic studies and mutational analyses to identify the heme iron ligands in yeast <i>iso</i>-1 Met-SO cyt <i>c</i>. Similar to the alkaline form of native cyt <i>c</i>, 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 the 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 <i>c</i>. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K^#) and Met-SO is more than 3 orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt <i>c</i>. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt <i>c</i> with Co­(terpy)₂²⁺. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for nonswitchable M80A and M80K^#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt <i>c</i> (p<i>K</i>_a = 6.3 ± 0.1), increasing the intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt <i>c</i> specifically in apoptotic cells

Efficient Enhancement of the Visible-Light Absorption of Cyclometalated Ir(III) Complexes Triplet Photosensitizers with Bodipy and Applications in Photooxidation and Triplet–Triplet Annihilation Upconversion

We report molecular designing strategies to enhance the effective visible-light absorption of cyclometalated Ir­(III) complexes. Cationic cyclometalated Ir­(III) complexes were prepared in which boron–dipyrromethene (Bodipy) units were attached to the 2,2′-bipyridine (bpy) ligand via −CC– bonds at either the <i>meso</i>-phenyl (<b>Ir-2</b>) or 2 position of the π core of Bodipy (<b>Ir-3</b>). For the first time the effect of π conjugating (<b>Ir-3</b>) or tethering (<b>Ir-2</b>) of a light-harvesting chromophore to the coordination center on the photophysical properties was compared in detail. Ir­(ppy)₂(bpy) (<b>Ir-1</b>; ppy = 2-phenylpyridine) was used as model complex, which gives the typical weak absorption in visible range (ε < 4790 M^–1 cm^–1 in region > 400 nm). <b>Ir-2</b> and <b>Ir-3</b> showed much stronger absorption in the visible range (ε = 71 400 M^–1 cm^–1 at 499 nm and 83 000 M^–1 cm^–1 at 527 nm, respectively). Room-temperature phosphorescence was only observed for <b>Ir-1</b> (λ_em = 590 nm) and <b>Ir-3</b> (λ_em = 742 nm). <b>Ir-3</b> gives RT phosphorescence of the Bodipy unit. On the basis of the 77 K emission spectra, nanosecond transient absorption spectra, and spin density analysis, we proposed that Bodipy-localized long-lived triplet excited states were populated for <b>Ir-2</b> (τ_T = 23.7 μs) and <b>Ir-3</b> (87.2 μs). <b>Ir-1</b> gives a much shorter triplet-state lifetime (0.35 μs). Complexes were used as singlet oxygen (¹O₂) photosensitizers in photooxidation. The ¹O₂ quantum yield of <b>Ir-3</b> (Φ_Δ = 0.97) is ca. 2-fold of <b>Ir-2</b> (Φ_Δ = 0.52). Complexes were also used as triplet photosensitizer for TTA upconversion; upconversion quantum yields of 1.2% and 2.8% were observed for <b>Ir-2</b> and <b>Ir-3</b>, respectively. Our results proved that the strong absorption of visible light of <b>Ir-2</b> failed to enhance production of a triplet excited state. These results are useful for designing transition metal complexes that show <i>effective</i> strong visible-light absorption and long-lived triplet excited states, which can be used as ideal triplet photosensitizers in photocatalysis and TTA upconversion

Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins

Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model to elucidate roles of the interdomain interface on properties of iron centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with the full-length diheme c4 and its Met-to-His ligand variants, were characterized by a variety of spectroscopic and stability measurements. In both isolated domains, the heme iron is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains, and isolated c4-A is folded. In contrast, the isolated c4-B is partially unfolded, and the interface with c4-A guides folding of this domain. The c4-A and c4-B domains have the propensity to interact even without the polypeptide linker. Thermodynamic cycles have revealed properties of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by the interface. This study illustrates the effects of the interface on tuning structural and redox properties of multiheme proteins and enriches our understanding of redox-dependent complexation

A homogenously stained case of Ki67.

<p>With a homogenous diffuse high Ki67 LI across the slide.</p

The ICC between VA and DIA of Ki67 LI, stratified by paired-difference between VA values of hot-spot score and average score.

<p>The ICC between VA and DIA of Ki67 LI, stratified by paired-difference between VA values of hot-spot score and average score.</p

A score area was analyzed by DIA.

<p>Positive tumor cells were labeled with red dots and negative tumor cells with green dots. The areas surrounded by black lines were excluded. A few negative tumor cells weren’t recognized (black arrow).</p

A heterogeneously stained case of Ki67.

<p>With a high Ki67 LI on the left and a low Ki67 LI on the right.</p

A selected area was to be analyzed by DIA.

<p>The scored area was surrounded by green lines and the mesenchymal components were excluded by black lines.</p

The median of paired-difference between VA and DIA values of Ki67 LI.

<p>The median of paired-difference between VA and DIA values of Ki67 LI.</p

The ICC between VA and DIA of Ki67 LI in G2-G3, ER positive /HER2 negative cases according to two score methods.

<p>The ICC between VA and DIA of Ki67 LI in G2-G3, ER positive /HER2 negative cases according to two score methods.</p