Multimerization of Solution-State Proteins by Tetrakis(4-sulfonatophenyl)porphyrin

Surface binding and interactions of anionic porphyins bound to cationic proteins have been studied for nearly three decades and are relevant as models for protein surface molecular recognition and photoinitiated electron transfer. However, interpretation of data in nearly all reports explicitly or implicitly assumed interaction of porphyrin with monodisperse proteins in solutions. In this report, using small-angle X-ray scattering with solution phase samples, we demonstrate that horse heart cytochrome (cyt) <i>c</i>, triheme cytochrome <i>c</i>₇ PpcA from <i>Geobacter sulfurreducens</i>, and hen egg lysozyme multimerize in the presence of zinc tetrakis­(4-sulfonatophenyl)­porphyrin (ZnTPPS). Multimerization of cyt <i>c</i> showed a pH dependence with a stronger apparent binding affinity under alkaline conditions and was weakened in the presence of a high salt concentration. Ferric-cyt <i>c</i> formed complexes larger than those formed by ferro-cyt c. Free base TPPS and FeTPPS facilitated formation of complexes larger than those of ZnTPPS. No increase in protein aggregation state for cationic proteins was observed in the presence of cationic porphyrins. All-atom molecular dynamics simulations of cyt <i>c</i> and PpcA with free base TPPS corroborated X-ray scattering results and revealed a mechanism by which the tetrasubstituted charged porphyrins serve as bridging ligands nucleating multimerization of the complementarily charged protein. The final aggregation products suggest that multimerization involves a combination of electrostatic and hydrophobic interactions. The results demonstrate an overlooked complexity in the design of multifunctional ligands for protein surface recognition

Zinc-Catalyzed Two-Electron Nickel(IV/II) Redox Couple for Multi-Electron Storage in Redox Flow Batteries

Energy storage is a vital aspect for the successful implementation of renewable energy resources on a global scale. Herein, we investigated the redox cycle of nickel(II) bis(diethyldithiocarbamate), NiII(dtc)2, for potential use as a multielectron storage catholyte in nonaqueous redox flow batteries (RFBs). Previous studies have shown that the unique redox cycle of NiII(dtc)2 offers 2e– chemistry upon oxidation from NiII → NiIV but 1e– chemistry upon reduction from NiIV → NiIII → NiII. Electrochemical experiments presented here show that the addition of as little as 10 mol % ZnII(ClO4)2 to the electrolyte consolidates the two 1e– reduction peaks into a single 2e– reduction where [NiIV(dtc)3]+ is reduced directly to NiII(dtc)2. This catalytic enhancement is believed to be due to ZnII removal of a dtc– ligand from a NiIII(dtc)3 intermediate, resulting in more facile reduction to NiII(dtc)2. The addition of ZnII also improves the 2e– oxidation, shifting the anodic peak negative and decreasing the 2e– peak separation. H-cell cycling experiments showed that 97% Coulombic efficiency and 98% charge storage efficiency was maintained for 50 cycles over 25 h using 0.1 M ZnII(ClO4)2 as the supporting electrolyte. If ZnII(ClO4)2 was replaced with TBAPF6 in the electrolyte, the Coulombic efficiency fell to 78%. The use of ZnII to increase the reversibility of 2e– transfer is a promising result that points to the ability to use nickel dithiocarbonates for multielectron storage in RFBs

Electron Paramagnetic Resonance Characterization of the Triheme Cytochrome from <i>Geobacter sulfurreducens</i>

Periplasmic cytochrome A (PpcA) is a representative of a broad class of multiheme cytochromes functioning as protein “nanowires” for storage and extracellular transfer of multiple electrons in the δ-proteobacterium <i>Geobacter sulfurreducens</i>. PpcA contains three bis-His coordinated hemes held in a spatial arrangement that is highly conserved among the multiheme cytochromes c₃ and c₇ families, carries low potential hemes, and is notable for having one of the lowest number of amino acids utilized to maintain a characteristic protein fold and site-specific heme function. Low temperature X-band electron paramagnetic resonance (EPR) spectroscopy has been used to characterize the electronic configuration of the Fe­(III) and the ligation mode for each heme. The three sets of EPR signals are assigned to individual hemes in the three-dimensional crystal structure. The relative energy levels of the Fe­(III) 3d orbitals for individual hemes were estimated from the principal <i>g</i>-values. The observed <i>g</i>-tensor anisotropy was used as a probe of electronic structure of each heme, and differences were determined by specifics of axial ligation. To ensure unambiguous assignment of highly anisotropic low-spin (HALS) signal to individual hemes, EPR analyses of iron atom electronic configurations have been supplemented with investigation of porphyrin macrocycles by one-dimensional ¹H NMR chemical shift patterns for the methyl substituents. Within optimized geometry of hemes in PpcA, the magnetic interactions between hemes were found to be minimal, similar to the c₃ family of tetraheme cytochromes

Macroscopic oxidation curves of PpcA mutants (black solid lines) and wild-type (red dashed line) at pH 7.5.

<p>The curves were calculated as a function of the solution reduction potential using the parameters listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105566#pone-0105566-t002" target="_blank">Table 2</a>. The midpoint reduction potentials of proteins (</p><p>E_app</p>) are indicated in the inset.<p></p

Thermodynamic parameters for PpcA mutants.

<p>For comparison the values previously obtained for PpcA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105566#pone.0105566-Morgado3" target="_blank">[17]</a> were also included. For each cytochrome, the fully reduced and protonated protein was taken as reference. Diagonal values (in bold) correspond to oxidation energies of the hemes and deprotonating energy of the redox-Bohr centre. Off-diagonal values are the redox (heme-heme) and redox-Bohr (heme-proton) interactions energies. All energies are reported in meV, with standard errors given in parenthesis.</p

Oxidized fractions of the individual hemes for PpcA mutants (solid lines) and wild-type (dashed lines) at pH 7.5.

<p>The curves were calculated as a function of the solution reduction potential using the parameters listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105566#pone-0105566-t002" target="_blank">Table 2</a>. The midpoint reduction potentials of the hemes (</p><p>e_app</p>) are also indicated.<p></p

Comparison of the results obtained from the thermodynamic characterization of PpcA mutants at pH 7.5.

<p>For comparison the values previously obtained for PpcA, PpcB, PpcD and PpcE <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105566#pone.0105566-Morgado3" target="_blank">[17]</a> and PpcAM58 mutants <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105566#pone.0105566-Morgado7" target="_blank">[29]</a> were also included. Δ<i>e_app</i> (2^nd/1^st) is the difference between the <i>e_app</i> values of the second and the first heme to oxidized. Δ<i>e_app</i> (3^rd/2^nd) is the difference between the <i>e_app</i> values of the third and second heme to oxidized.</p

Spatial location of lysine residues mutated in this work depicted in PpcA solution structure (PDB code, 2LDO [11]).

<p>The PpcA polypeptide chain (gray) is shown as C_α ribbon and heme groups (red). The side chain of Lys⁹, Lys¹⁸, Lys²² Lys⁴³, Lys⁵² and Lys⁶⁰ (green) are represented as stick drawings. The hemes are numbered I, III and IV, a designation that derives from the superimposition of the hemes in cytochromes <i>c</i>₇ with those of the structurally homologous tetraheme cytochromes <i>c</i>₃.</p

Distance between the C_β of selected lysine residues to the nearest heme iron(s) in the solution structure of PpcA (PDB code, 2LDO [11]).

<p>Distance between the C_β of selected lysine residues to the nearest heme iron(s) in the solution structure of PpcA (PDB code, 2LDO <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105566#pone.0105566-Morgado2" target="_blank">[11]</a>).</p

Oxidation fraction of K9, K18 and K22 PpcA mutants (solid symbols and lines) and PpcA (open symbols and dashed lines) at pH 6 and pH 8.

<p>Data for hemes I, III and IV are colored in green, orange and blue, respectively. The heme oxidation fractions were calculated according to equation </p><p>x_i</p>  =  (δ_i-δ₀)/(δ₃-δ₀), where δ_i, δ₀, and δ₃ are the observed chemical shifts of each methyl in stage i, 0, and 3, respectively.<p></p