The Birch reduction is a widely used synthetic tool to reduce arenes to 1,4-cyclohexadienes. Its harsh cryogenic reaction conditions and the dependence on alkali metals have motivated researchers to explore alternative approaches. In anaerobic aromatic compound degrading microbes, class II benzoyl-coenzyme A (CoA) reductases (BCRs) reduce benzoyl-CoA to the conjugated cyclohexa-1,5-diene-1-carboxyl-CoA (1,5-dienoyl-CoA) at a tungsten-bis-metallopterin (MPT) cofactor. Though previous structure-based computational studies were in favor of a Birch-like reduction via W(V)/radical intermediates, any experimental evidence for such a mechanism was lacking. Here, we combined freeze-quench and equilibrium electron paramagnetic resonance (EPR) spectroscopic analyses in H2O, D2O, and H217O with redox titrations using wild-type and molecular variants of the catalytic BamB subunit of class II BCR from the anaerobic bacterium Geobacter metallireducens. We provide spectroscopic evidence for a kinetically competent radical/W(V)-OH intermediate obtained after hydrogen atom transfer from the W-aqua-ligand to the aromatic ring and for an invariant histidine as a proton donor assisting the second electron transfer. Quantum mechanical/molecular mechanical calculations suggest that the unique tetrahydro state of both pyranopterins is essential for the reversibility of enzymatic Birch reduction. This work elucidates nature's solution for the chemically demanding Birch reduction and demonstrates how the reactivity of MPT cofactors can be expanded to highly challenging radical chemistry at the negative limit of the biological redox window.

We used a series of modified/substituted GGH analogues to investigate the kinetics of Cu(ii) binding to ACTUN peptides. Rules for rate modulation by 1^st and 2^nd sphere interactions were established, providing crucial insight into elucidation of the reaction mechanism and its contribution to biological copper transport.

The heme enzyme chlorite dismutase (Cld) catalyzes O-O bond formation as part of the conversion of the toxic chlorite (ClO2-) to chloride (Cl-) and molecular oxygen (O2). Enzymatic O-O bond formation is rare in nature, and therefore, the reaction mechanism of Cld is of great interest. Microsecond timescale pre-steady-state kinetic experiments employing Cld from Azospira oryzae (AoCld), the natural substrate chlorite, and the model substrate peracetic acid (PAA) reveal the formation of distinct intermediates. AoCld forms a complex with PAA rapidly, which is cleaved heterolytically to yield Compound I, which is sequentially converted to Compound II. In the presence of chlorite, AoCld forms an initial intermediate with spectroscopic characteristics of a 6-coordinate high-spin ferric substrate adduct, which subsequently transforms at kobs = 2-5 × 104 s-1 to an intermediate 5-coordinated high-spin ferric species. Microsecond-Timescale freeze-hyperquench experiments uncovered the presence of a transient low-spin ferric species and a triplet species attributed to two weakly coupled amino acid cation radicals. The intermediates of the chlorite reaction were not observed with the model substrate PAA. These findings demonstrate the nature of physiologically relevant catalytic intermediates and show that the commonly used model substrate may not behave as expected, which demands a revision of the currently proposed mechanism of Clds. The transient triplet-state biradical species that we designate as Compound T is, to the best of our knowledge, unique in heme enzymology. The results highlight electron paramagnetic resonance spectroscopic evidence for transient intermediate formation during the reaction of AoCld with its natural substrate chlorite. In the proposed mechanism, the heme iron remains ferric throughout the catalytic cycle, which may minimize the heme moiety's reorganization and thereby maximize the enzyme's catalytic efficiency.

The amino-terminal copper and nickel/N-terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards Cu^II ions and have been implicated in human copper physiology. Little is known, however, about the rate and exact mechanism of formation of such complexes. We used the stopped-flow and microsecond freeze-hyperquenching (MHQ) techniques supported by steady-state spectroscopic and electrochemical data to demonstrate the formation of partially coordinated intermediate Cu^II complexes formed by glycyl–glycyl–histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two-nitrogen coordination, t_1/2≈100 ms at pH 6.0 and the ability to maintain the Cu^II/Cu^I redox pair is the best candidate for the long-sought reactive species in extracellular copper transport.

Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO₂⁻ (chlorite) to innocuous Cl⁻ and O₂. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Graphic abstract: Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between Fe^III and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.[Figure not available: see fulltext.]

In the original article published, in the g_y value (column) of the H₂O/OH⁻species (row) of Table 2 was mistakenly given as “1.18” and the correct value is “2.18”.

Abstract: Chlorite dismutase is a unique heme enzyme that catalyzes the conversion of chlorite to chloride and molecular oxygen. The enzyme is highly specific for chlorite but has been known to bind several anionic and neutral ligands to the heme iron. In a pH study, the enzyme changed color from red to green in acetate buffer pH 5.0. The cause of this color change was uncovered using UV–visible and EPR spectroscopy. Chlorite dismutase in the presence of acetate showed a change of the UV–visible spectrum: a redshift and hyperchromicity of the Soret band from 391 to 404 nm and a blueshift of the charge transfer band CT1 from 647 to 626 nm. Equilibrium binding titrations with acetate resulted in a dissociation constant of circa 20 mM at pH 5.0 and 5.8. EPR spectroscopy showed that the acetate bound form of the enzyme remained high spin S = 5/2, however with an apparent change of the rhombicity and line broadening of the spectrum. Mutagenesis of the proximal arginine Arg183 to alanine resulted in the loss of the ability to bind acetate. Acetate was discovered as a novel ligand to chlorite dismutase, with evidence of direct binding to the heme iron. The green color is caused by a blueshift of the CT1 band that is characteristic of the high spin ferric state of the enzyme. Any weak field ligand that binds directly to the heme center may show the red to green color change, as was indeed the case for fluoride.

The oxygen-independent nitrate-nitrite-nitric oxide (NO) pathway is considered as a substantial source of NO in mammals. Dietary nitrate/nitrite are distributed throughout the body and reduced to NO by the action of various enzymes. The intermembrane spaced (IMS), molybdenum cofactor-dependent sulfite oxidase (SO) was shown to catalyze such a nitrite reduction. In this study we asked whether the primary function of SO – sulfite oxidation – and its novel function – nitrite reduction – impact each other. First, we utilized benzyl viologen as artificial electron donor to investigate steady state NO synthesis by SO and found fast (k _cat = 14 s ⁻¹ ) nitrite reduction of SO full-length and its isolated molybdenum domain at pH 6.5. Next, we determined the impact of nitrite on pre-steady state kinetics in SO catalysis and identified nitrite as a pH-dependent inhibitor of SO reductive and oxidative half reaction. Finally, we report on the time-dependent formation of the paramagnetic Mo(V) species following nitrite reduction and demonstrate that sulfite inhibits nitrite reduction. In conclusion, we propose a pH-dependent reciprocal regulation of sulfite oxidation and nitrite reduction by each substrate, thus facilitating quick responses to hypoxia induced changes in the IMS, which may function in protecting the cell from reactive oxygen species production.

The study of the structure, function, folding and conformational transitions of cytochrome c is of great interest because this protein plays an important role in biological electron transport and apoptosis. The different native and non-native conformations have been studied extensively under equilibrium conditions at different pH values, however, kinetic studies are rare because they require technically challenging rapid mixing and spectroscopic monitoring techniques. Here we present the refolding kinetics of acid denatured cytochrome c using the pH jump technique from pH 2 to pH 4.7 in combination with a new ultrafast continuous flow mixing device that allows time resolved measurements to the microsecond time scale. Our results show that the initial refolding of denatured oxidized cytochrome c occurs very rapidly with a time constant τ = 10 μs, and is followed by discrete refolding steps with time constants of 56 and 208 μs. Electron paramagnetic resonance analysis of the different intermediates, obtained by microsecond freeze hyper quenching showed that the first two intermediates are predominantly high spin, and the third intermediate is the low spin species with complete His/Met coordination. The initial rapid phase is characterized by the formation of high spin species distinct from the completely unfolded state. We interpret this as the formation of a five coordinate species with His18 as the axial ligand or six coordinate with water and His18 as the axial ligands.

To afford mechanistic studies in enzyme kinetics and protein folding in the microsecond time domain we have developed a continuous-flow microsecond time-scale mixing instrument with an unprecedented dead-time of 3.8 ± 0.3 μs. The instrument employs a micro-mixer with a mixing time of 2.7 μs integrated with a 30 mm long flow-cell of 109 μm optical path length constructed from two parallel sheets of silver foil; it produces ultraviolet-visible spectra that are linear in absorbance up to 3.5 with a spectral resolution of 0.4 nm. Each spectrum corresponds to a different reaction time determined by the distance from the mixer outlet, and by the fluid flow rate. The reaction progress is monitored in steps of 0.35 μs for a total duration of ~600 μs. As a proof of principle the instrument was used to study spontaneous protein refolding of pH-denatured cytochrome c. Three folding intermediates were determined: after a novel, extremely rapid initial phase with τ = 4.7 μs, presumably reflecting histidine re-binding to the iron, refolding proceeds with time constants of 83 μs and 345 μs to a coordinatively saturated low-spin iron form in quasi steady state. The time-resolution specifications of our spectrometer for the first time open up the general possibility for comparison of real data and molecular dynamics calculations of biomacromolecules on overlapping time scales.

Soluble quinoprotein (PQQ-containing) glucose dehydrogenase (sGDH, EC 1.1.99.35) catalyzes the oxidation of β-d-glucose to d-glucono-δ-lactone. Although sGDH has many analytical applications, the relationship between activity and substrate concentration is not well established. Previous steady-state kinetic studies revealed a negative cooperativity effect which has recently been ascribed to subunit interaction. To investigate this conclusion, stopped-flow kinetic experiments were carried out on the reaction in which oxidized enzyme (E_ox) was reduced with substrates to E_red. The appearance of E_red is observed to be preceded by formation of an intermediate enzyme form, Int, which is mono-exponentially formed from E_ox. However, the rate of conversion of Int into E_red depends hyperbolically on the concentration of substrate (leading to a 35-fold stimulation in the case of glucose). Evidence is provided that substrate not only binds to E_ox but also to Int and E_red as well, and that the binding to Int causes the significant stimulation of Int decay. It is proposed that a proton shuffling step is involved in the decay, which is facilitated by binding of substrate to Int. Substituting the PQQ-activating Ca by a Ba ion lowered all reaction rates but did not change the stimulation factor. In summary, the previous proposal that the cooperativity effect of sGDH is due to interaction between its substrate-loaded subunits is incorrect; it is due to substrate-assisted catalysis of the enzyme. Enzymes: EC 1.1.99.35 – soluble quinoprotein glucose dehydrogenase.