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.

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 k obs = 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.

Unexpected photosensitivity of the well-characterized heme enzyme chlorite dismutase.

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. 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 FeIII 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.

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

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".

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.

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.

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 (kcat = 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.

Refolding kinetics of cytochrome c studied with microsecond timescale continuous-flow UV-vis spectroscopy and rapid freeze-quench EPR.

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.

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.

The cooperativity effect in the reaction of soluble quinoprotein (PQQ-containing) glucose dehydrogenase is not due to subunit interaction but to substrate-assisted catalysis.

Soluble quinoprotein (PQQ-containing) glucose dehydrogenase (sGDH, EC 1.1.99.35) catalyzes the oxidation of ß-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 (Eox ) was reduced with substrates to Ered . The appearance of Ered is observed to be preceded by formation of an intermediate enzyme form, Int, which is mono-exponentially formed from Eox . However, the rate of conversion of Int into Ered 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 Eox but also to Int and Ered 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.

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.

Adaptation to a high-tungsten environment: Pyrobaculum aerophilum contains an active tungsten nitrate reductase.

Nitrate reductases (Nars) belong to the DMSO reductase family of molybdoenzymes. The hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum exhibits nitrate reductase (Nar) activity even at WO(4)(2-) concentrations that are inhibitory to bacterial Nars. In this report, we establish that the enzyme purified from cells grown with 4.5 µM WO(4)(2-) contains W as the metal cofactor but is otherwise identical to the Mo-Nar previously purified from P. aerophilum grown at low WO(4)(2-) concentrations. W is coordinated by a bis-molybdopterin guanine dinucleotide cofactor. The W-Nar has a 2-fold lower turnover number (633 s(-1)) but the same K(m) value for nitrate (56 µM) as the Mo-Nar. Quinol reduction and nitrate oxidation experiments monitored by EPR with both pure W-Nar and mixed W- and Mo-Nar preparations suggest a monodentate ligation by the conserved Asp241 for W(V), while Asp241 acts as a bidentate ligand for Mo(V). Redox titrations of the Mo-Nar revealed a midpoint potential of 88 mV for Mo(V/IV). The E(m) for W(V/IV) of the purified W-Nar was estimated to be -8 mV. This relatively small difference in midpoint potential is consistent with comparable enzyme activities of W- and Mo-Nars. Unlike bacterial Nars, the P. aerophilum Nar contains a unique membrane anchor, NarM, with a single heme of the o(P) type (E(m) = 126 mV). In contrast to bacterial Nars, the P. aerophilum Nar faces the cell's exterior and, hence, does not contribute to the proton motive force. Formate is used as a physiological electron donor. This is the first description of an active W-containing Nar demonstrating the unique ability of hyperthermophiles to adapt to their high-WO(4)(2-) environment.

Very early reaction intermediates detected by microsecond time scale kinetics of cytochrome cd1-catalyzed reduction of nitrite.

Paracoccus pantotrophus cytochrome cd(1) is a nitrite reductase found in the periplasm of many denitrifying bacteria. It catalyzes the reduction of nitrite to nitric oxide during the denitrification part of the biological nitrogen cycle. Previous studies of early millisecond intermediates in the nitrite reduction reaction have shown, by comparison with pH 7.0, that at the optimum pH, approximately pH 6, the earliest intermediates were lost in the dead time of the instrument. Access to early time points (approximately 100 micros) through use of an ultra-rapid mixing device has identified a spectroscopically novel intermediate, assigned as the Michaelis complex, formed from reaction of fully reduced enzyme with nitrite. Spectroscopic observation of the subsequent transformation of this species has provided data that demand reappraisal of the general belief that the two subunits of the enzyme function independently.

Purification and characterization of the MQH2:NO oxidoreductase from the hyperthermophilic archaeon Pyrobaculum aerophilum.

The membrane-bound NO reductase from the hyperthermophilic denitrifying archaeon Pyrobaculum aerophilum was purified to homogeneity. The enzyme displays MQH2:NO oxidoreductase (qNOR) activity, consists of a single subunit, and contains heme and nonheme iron in a 2:1 ratio. The combined results of EPR, resonance Raman, and UV-visible spectroscopy show that one of the hemes is bis-His-coordinated low spin (gz = 3.015; gy = 2.226; gx = 1.45), whereas the other heme adopts a high spin configuration. The enzyme also contains one nonheme iron center, which in the oxidized enzyme is antiferromagnetically coupled to the high spin heme. This binuclear high spin heme/nonheme iron center is EPR-silent and the site of NO reduction. The reduced high spin heme is bound to a neutral histidine and can bind CO to form of a low spin complex. The oxidized high spin heme binds NO, yielding a ferric nitrosyl complex, the intermediate causing the commonly found substrate inhibition in NO reductases (Ki(NO) = 7 microm). The qNOR as present in the membrane is, in contrast to the purified enzyme, quite thermostable, incubation at 100 degrees C for 86 min leading to 50% inhibition. The pure enzyme lacks heme b and instead contains stoichiometric amounts of hemes Op1 and Op2, ethenylgeranylgeranyl and hydroxyethylgeranylgeranyl derivatives of heme b, respectively. The archaeal qNOR is the first example of a NO reductase, which contains modified hemes reminiscent of cytochrome bo3 and aa3 oxidases. This report is the first describing the purification and structural and spectroscopic properties of a thermostable NO reductase.