Die Elektronentunnelraten im Atmungskettenkomplex I sind auf eine effiziente Energiewandlung abgestimmt

Der Atmungskettenkomplex I wandelt die freie Energie, die bei der Reduktion von Ubichinon durch NADH frei wird, in einen Protonengradienten über die Membran um. Die biologische Redoxreaktion wird von einem Flavinmononukleotid und einer Kette von sieben Eisen-Schwefel-Zentren katalysiert. Die Elektronentransfergeschwindigkeiten zwischen den Zentren wurden über eine schnelle \u84Freeze-Quench“-Methode und Analyse der Proben mittels EPR- und UV/Vis-Spektroskopie bestimmt. Der Komplex I oxidiert sehr schnell drei Moleküle NADH; dabei hängt die Elektronentunnelrate zwischen den beiden Zentren der Kette mit dem grçßten Abstand zueinander vom Redoxzustand des distalen Zentrums N2 ab. Diese Geschwindigkeit ist sechsmal geringer, wenn N2 im reduzierten Zustand vorliegt. Die konformativen Änderungen, die mit der Reduktion des Zentrums N2 einhergehen, verlangsamen die elektronische Kopplung des längsten Transferschritts. Die Kette der Eisen-Schwefel-Zentren ist somit nicht einfach ein Draht für den Elektronentransfer, sondern sie passt die Elektronentunnelraten der Zeitskala der konformativen Bewegungen an, die für die Protonentranslokation bençtigt werden. Die Synchronisierung von Geschwindigkeiten ist ein generelles Prinzip, um die Spezifität enzymatischer Reaktionen zu erhçhen.BT/BiotechnologyApplied Science

Electron Tunneling Rates in Respiratory Complex?I Are Tuned for Efficient Energy Conversion

Respiratory complex I converts the free energy of ubiquinone reduction by NADH into a proton motive force, a redox reaction catalyzed by flavin mononucleotide(FMN) and a chain of seven iron–sulfur centers. Electron transfer rates between the centers were determined by ultrafast freeze-quenching and analysis by EPR and UV/Vis spectroscopy. The complex rapidly oxidizes three NADH molecules. The electron-tunneling rate between the most distant centers in the middle of the chain depends on the redox state of center N2 at the end of the chain, and is sixfold slower when N2 is reduced. The conformational changes that accompany reduction of N2 decrease the electronic coupling of the longest electron-tunneling step. The chain of iron–sulfur centers is not just a simple electron-conducting wire; it regulates the electron-tunneling rate synchronizing it with conformation-mediated proton pumping, enabling efficient energy conversion. Synchronization of rates is a principle means of enhancing the specificity of enzymatic reactions.BT/BiotechnologyApplied Science

Microsecond time-scale kinetics of transient biochemical reactions

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.BT/Biocatalysi

The rates of Cu(ii)-ATCUN complex formation. Why so slow?

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 1st and 2nd sphere interactions were established, providing crucial insight into elucidation of the reaction mechanism and its contribution to biological copper transport.Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi

Correction to: A traffic light enzyme: acetate binding reversibly switches chlorite dismutase from a red- to a green-colored heme protein (JBIC Journal of Biological Inorganic Chemistry, (2020), 25, 4, (609-620), 10.1007/s00775-020-01784-1)

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

Unique Biradical Intermediate in the Mechanism of the Heme Enzyme Chlorite Dismutase

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. BT/Biocatalysi

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

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.BT/Biocatalysi

Enzymatic Birch Reduction via Hydrogen Atom Transfer at an Aqua-Tungsten- bis-Metallopterin Cofactor

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. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public.BT/Biocatalysi

Correction to: A traffic light enzyme: acetate binding reversibly switches chlorite dismutase from a red- to a green-colored heme protein (JBIC Journal of Biological Inorganic Chemistry, (2020), 25, 4, (609-620), 10.1007/s00775-020-01784-1)

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