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Background & Aims Porphyrias are caused by porphyrin accumulation resulting from defects in the heme biosynthetic pathway that typically lead to photosensitivity and possible end-stage liver disease with an increased risk of hepatocellular carcinoma. Our aims were to study the mechanism of porphyrin-induced cell damage and protein aggregation, including liver injury, where light exposure is absent. Methods Porphyria was induced in vivo in mice using 3,5-diethoxycarbonyl-1,4-dihydrocollidine or in vitro by exposing human liver Huh7 cells and keratinocytes, or their lysates, to protoporphyrin-IX, other porphyrins, or to δ-aminolevulinic acid plus deferoxamine. The livers, cultured cells, or porphyrin exposed purified proteins were analyzed for protein aggregation and oxidation using immunoblotting, mass spectrometry, and electron paramagnetic resonance spectroscopy. Consequences on cell-cycle progression were assessed. Results Porphyrin-mediated protein aggregation required porphyrin-photosensitized singlet oxygen and porphyrin carboxylate side-chain deprotonation, and occurred with site-selective native protein methionine oxidation. Noncovalent interaction of protoporphyrin-IX with oxidized proteins led to protein aggregation that was reversed by incubation with acidified n-butanol or high-salt buffer. Phototoxicity and the ensuing proteotoxicity, mimicking porphyria photosensitivity conditions, were validated in cultured keratinocytes. Protoporphyrin-IX inhibited proteasome function by aggregating several proteasomal subunits, and caused cell growth arrest and aggregation of key cell proliferation proteins. Light-independent synergy of protein aggregation was observed when porphyrin was applied together with glucose oxidase as a secondary peroxide source. Conclusions Photo-excitable porphyrins with deprotonated carboxylates mediate protein aggregation. Porphyrin-mediated proteotoxicity in the absence of light, as in the liver, requires porphyrin accumulation coupled with a second tissue oxidative injury. These findings provide a potential mechanism for internal organ damage and photosensitivity in porphyrias., Graphical abstract

Cytochrome c peroxidases (bCcPs) are diheme enzymes required for the reduction of H2O2 to water in bacteria. There are two classes of bCcPs: one is active in the diferric form (constitutively active), and the other requires the reduction of the high-potential heme (H-heme) before catalysis commences (reductively activated) at the low-potential heme (L-heme). To improve our understanding of the mechanisms and heme electronic structures of these different bCcPs, a constitutively active bCcP from Nitrosomonas europaea ( NeCcP) and a reductively activated bCcP from Shewanella oneidensis ( SoCcP) were characterized in both the diferric and semireduced states by electron paramagnetic resonance (EPR), resonance Raman (rRaman), and magnetic circular dichroism (MCD) spectroscopy. In contrast to some previous crystallographic studies, EPR and rRaman spectra do not indicate the presence of significant amounts of a five-coordinate, high-spin ferric heme in NeCcP or SoCcP in either the diferric or semireduced state in solution. This observation points toward a mechanism of activation in which the active site L-heme is not in a static, five-coordinate state but where the activation is more subtle and likely involves formation of a six-coordinate hydroxo complex, which could then react with hydrogen peroxide in an acid-base-type reaction to create Compound 0, the ferric hydroperoxo complex. This mechanism lies in stark contrast to the diheme enzyme MauG that exhibits a static, five-coordinate open heme site at the peroxidatic heme and that forms a more stable FeIV═O intermediate.

Myoglobin reconstituted with iron porphycene catalyzes the cyclopropanation of styrene with ethyl diazoacetate. Compared to native myoglobin, the reconstituted protein significantly accelerates the catalytic reaction and the kcat/Km value is 26-fold enhanced. Mechanistic studies indicate that the reaction of the reconstituted protein with ethyl diazoacetate is 615-fold faster than that of native myoglobin. The metallocarbene species reacts with styrene with the apparent second-order kinetic constant of 28 mM–1 s–1 at 25 °C. Complementary theoretical studies support efficient carbene formation by the reconstituted protein that results from the strong ligand field of the porphycene and fewer intersystem crossing steps relative to the native protein. From these findings, the substitution of the cofactor with an appropriate metal complex serves as an effective way to generate a new biocatalyst.

Methyl-coenzyme M reductase, the rate-limiting enzyme in methanogenesis and anaerobic methane oxidation, is responsible for the biological production of more than 1 billion tons of methane per year. The mechanism of methane synthesis is thought to involve either methyl-nickel(III) or methyl radical/Ni(II)-thiolate intermediates. We employed transient kinetic, spectroscopic, and computational approaches to study the reaction between the active Ni(I) enzyme and substrates. Consistent with the methyl radical-based mechanism, there was no evidence for a methyl-Ni(III) species; furthermore, magnetic circular dichroism spectroscopy identified the Ni(II)-thiolate intermediate. Temperature-dependent transient kinetics also closely matched density functional theory predictions of the methyl radical mechanism. Identifying the key intermediate in methanogenesis provides fundamental insights to develop better catalysts for producing and activating an important fuel and potent greenhouse gas.

The small, stable heme protein myoglobin (Mb) was modified through cofactor substitution and mutagenesis to develop a new catalyst for carbene transfer reactions. The native heme was removed from wild-type Mb and several Mb His64 mutants (H64D, H64A, H64V), and the resulting apoproteins were reconstituted with ruthenium mesoporphyrin IX (RuMpIX). The reconstituted proteins (RuMb) were characterized by UV-vis and circular dichroism spectroscopy and were used as catalysts for the N-H insertion of aniline derivatives and the cyclopropanation of styrene derivatives. The best catalysts for each reaction were able to achieve turnover numbers (TON) up to 520 for the N-H insertion of aniline, and 350 TON for the cyclopropanation of vinyl anisole. Our results show that RuMb is an effective catalyst for N-H insertion, with the potential to further increase the activity and stereoselectivity of the catalyst in future studies. Compared to native Mb ("FeMb"), RuMb is a more active catalyst for carbene transfer reactions, which leads to both heme and protein modification and degradation and, hence, to an overall much-reduced lifetime of the catalyst. This leads to lower TONs for RuMb compared to the iron-containing analogues. Strategies to overcome this limitation are discussed. Finally, comparison is also made to FeH64DMb and FeH64AMb, which have not been previously investigated for carbene transfer reactions.

Pentaammineruthenium(III) complexes of deoxyinosine (dIno) and xanthosine (Xao) ([RuIII(NH3)5(L)], L is dIno, Xao) in basic solution were studied by UV–vis spectroscopy, liquid chromatography/electrospray ionization mass spectrometry, and high-performance liquid chromatography. Both RuIII complexes disproportionate to RuII and RuIV. Disproportionation followed the rate law d[RuII]/dt = (k o + k 1[OH−])[RuIII]. k o and k 1 of disproportionation at 25 °C were 2.1 (±0.1) × 10−3 s−1 and 21.4 ± 3.2 M−1 s−1, respectively, for [RuIII(NH3)5(dIno)], and 3.5 (±0.7) × 10−4 s−1 and 59.7 ± 3.6 M−1 s−1, respectively, for [RuIII(NH3)5(Xao)]. The [RuIII(NH3)5(Xao)] complex disproportionates at a faster rate than [RuIII(NH3)5(dIno)] owing to the stronger electron-withdrawing effect of exocyclic oxygen in Xao. The activation parameters ΔH ‡ and ΔS ‡ for k 1 of [RuIII(NH3)5(dIno)] were 80.2 ± 15.2 kJ mol−1 and 47.6 ± 9.8 J K−1 mol−1, respectively, indicating that the disproportionation of RuIII to RuII and RuIV is favored owing to the positive entropy of activation. The final products of both complexes in basic solution under Ar were compared with those under O2. Under both conditions [Ru(NH3)5(8-oxo-L)] was produced, but via different mechanisms. In both aerobic and anaerobic conditions, the deprotonation of highly positively polarized C8-H of Ru-L by OH− initiates a two-electron redox reaction. For the next step, we propose a one-step two-electron redox reaction between L and RuIV under anaerobic conditions, which differentiates from Clarke’s mechanism of two consecutive one-electron redox reactions between L, RuIII, and O2.

Among the many mechanisms for the oxidation of guanine derivatives (G) assisted by transition metals, Ru(III) and Pt(IV) metal ions share basically the same principle. Both Ru(III)- and Pt(IV)-bound G have highly positively polarized C8-H's that are susceptible to deprotonation by OH(-), and both undergo two-electron redox reactions. The main difference is that, unlike Pt(IV), Ru(III) is thought to require O(2) to undergo such a reaction. In this study, however, we report that [Ru(III)(NH(3))(5)(dGuo)] (dGuo = deoxyguanosine) yields cyclic-5'-O-C8-dGuo (a two-electron G oxidized product, cyclic-dGuo) without O(2). In the presence of O(2), 8-oxo-dGuo and cyclic-dGuo were observed. Both [Ru(II)(NH(3))(5)(dGuo)] and cyclic-dGuo were produced from [Ru(III)(NH(3))(5)(dGuo)] accelerated by [OH(-)]. We propose that [Ru(III)(NH(3))(5)(dGuo)] disproportionates to [Ru(II)(NH(3))(5)(dGuo)] and [Ru(IV)(NH(3))(4)(NH(2)(-))(dGuo)], followed by a 5'-OH attack on C8 in [Ru(IV)(NH(3))(4)(NH(2)(-))(dGuo)] to initiate an intramolecular two-electron transfer from dGuo to Ru(IV), generating cyclic-dGuo and Ru(II) without involving O(2).

Nature's wisdom in enzyme design: Compounds I and II in the catalytic cycle of the Cytochrome P450 enzymes have been trapped and characterized recently. This work has provided further insight into the electronic structure and reactivity of these crucial intermediates, and key questions regarding the mechanism of these enzymes have finally been answered.

Nature's wisdom in enzyme design: Compounds I and II in the catalytic cycle of the Cytochrome P450 enzymes have been trapped and characterized recently. This work has provided further insight into the electronic structure and reactivity of these crucial intermediates, and key questions regarding the mechanism of these enzymes have finally been answered.

This review summarizes our current understanding of the geometric and electronic structures of ferrous and ferric heme–nitrosyls, which are of key importance for the biological functions and transformations of NO. In-depth correlations are made between these properties and the reactivities of these species. Here, a focus is put on the discoveries that have been made in the last 10 years, but previous findings are also included as necessary. Besides this, ferrous heme–nitroxyl complexes are also considered, which have become of increasing interest recently due to their roles as intermediates in NO and multiheme nitrite reductases, and because of the potential role of HNO as a signaling molecule in mammals. In recent years, computational methods have received more attention as a means of investigating enzyme reaction mechanisms, and some important findings from these theoretical studies are also highlighted in this chapter.