Reciprocal regulation of sulfite oxidation and nitrite reduction by mitochondrial sulfite oxidase

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 multitude of iron–sulfur clusters in respiratory complex I

Respiratory complex I couples the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. Complex I contains one non-covalently bound flavin mononucleotide and, depending on the species, up to ten iron–sulfur (Fe/S) clusters as cofactors. The reason for the presence of the multitude of Fe/S clusters in complex I remained enigmatic for a long time. The question was partly answered by investigations on the evolution of the complex revealing the stepwise construction of the electron transfer domain from several modules. Extension of the ancestral to the modern electron input domain was associated with the acquisition of several Fe/S-proteins. The X-ray structure of the complex showed that the NADH oxidation-site is connected with the quinone-reduction site by a chain of seven Fe/S-clusters. Fast enzyme kinetics revealed that this chain of Fe/S-clusters is used to regulate electron-tunneling rates within the complex. A possible function of the off-pathway cluster N1a is discussed. This article is part of a Special Issue entitled ‘EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2–6, 2016’, edited by Prof. Paolo Bernardi

Key Intermediate Species Reveal the Copper(II)‐Exchange Pathway in Biorelevant ATCUN/NTS Complexes

The amino-terminal copper and nickel/N-terminal site (ATCUN/NTS) present in proteins and bioactive peptides exhibits high affinity towards CuII 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 CuII complexes formed by glycyl-glycyl-histidine (GGH) peptide, the simplest ATCUN/NTS model. One of these novel intermediates, characterized by two-nitrogen coordination, t1/2 ≈100 ms at pH 6.0 and the ability to maintain the CuII /CuI redox pair is the best candidate for the long-sought reactive species in extracellular copper transport

Microsecond time-scale kinetics of transient biochemical reactions

<div><p>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 <i>c</i>. 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.</p></div

Design and geometry of the four-jet tangential ABAB micro-mixer.

<p>(a) Design of the micro-mixer body and the cuvette holder. (b) Geometry of the micro-mixer and the rectangular cuvette integrated in the plexiglass holder. Reaction components, A and B, enter the mixer body as indicated and subsequently flow through the four channels arranged in a cross. The components are then forced through a 100 μm wide and 50 μm deep μm Pt inlay mixing chamber (392.5 pL in volume and 3 mm in outer diameter). (c) Side-view slice through the mixer body and the Pt inlay along two opposite channels. (d) Top view of the channel arrangement and the premixing chamber of the dismounted mixer. The premixing chamber has dimensions of 100 x 100 x 50 μm³ yielding a volume of 500 pL.</p

Analysis of early stages in the re-folding of acid-denatured cytochrome <i>c</i>.

<p>Spectral components (a) and kinetic traces (b) of cytochrome <i>c</i> in a pH-jump (2 to 4.5) experiment obtained from singular value decomposition analysis. Time zero (t₀) at pH 2 (blue), I₁ (magenta), I₂ (red), I₃ (green). The spectrum of native protein (nat) at pH 6 (black) was obtained after prolonged incubation. (c) Interpretational scheme of the experiment as described in the text.</p

A traffic light enzyme: acetate binding reversibly switches chlorite dismutase from a red- to a green-colored heme protein

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