Microsecond freeze-hyperquenching: development of a new ultrafast micro-mixing and sampling technology and application to enzyme catalysis

A novel freeze-quench instrument with a characteristic «dead-time» of 137±18 μs is reported. The prototype has several key features that distinguish it from conventional freeze-quench devices and provide a significant improvement in time resolution: (a) high operating pressures (up to 400 bar) result in a sample flow with high linear rates (up to 200 m s⁻¹); (b) tangential micro-mixer with an operating volume of ∼1 nl yields short mixing times (up to 20 μs); (c) fast transport between the mixer and the cryomedium results in short reaction times: the ageing solution exits the mixer as a free-flowing jet, and the chemical reaction occurs “in-flight” on the way to the cryomedium; (d) a small jet diameter (∼20 μm) and a high jet velocity (∼200 m s⁻¹) provide high sample-cooling rates, resulting in a short cryofixation time (up to 30 μs). The dynamic range of the freeze-quench device is between 130 μs and 15 ms. The novel tangential micro-mixer efficiently mixes viscous aqueous solutions, showing more than 95% mixing at η≤4 (equivalent to protein concentrations up to 250 mg ml⁻¹), which makes it an excellent tool for the preparation of pre-steady state samples of concentrated protein solutions for spectroscopic structure analysis. The novel freeze-quench device is characterized using the reaction of binding of azide to metmyoglobin from horse heart. Reaction samples are analyzed using 77 K optical absorbance spectroscopy, and X-band EPR spectroscopy. A simple procedure of spectral analysis is reported that allows (a) to perform a quantitative analysis of the reaction kinetics and (b) to identify and characterize novel reaction intermediates. The reduction of dioxygen by the bo₃-type quinol oxidase from Escherichia coli is assayed using the MHQ technique. In these pilot experiments, low-temperature optical absorbance measurements show the rapid oxidation of heme o₃ in the first 137 μs of the reaction, accompanied by the formation of an oxo-ferryl species. X-band EPR spectroscopy shows that a short-living radical intermediate is formed during the oxidation of heme o₃. The radical decays within ∼1 ms concomitant with the oxidation of heme b, and can be attributed to the P_M reaction intermediate converting to the oxoferryl intermediate F. The general field of application of the freeze-quench methodology is discussed.     

Kinetic resolution of a tryptophan-radical intermediate in the reaction cycle of Paracoccus denitrificans cytochrome c oxidase

The catalytic mechanism, electron transfer coupled to proton pumping, of heme-copper oxidases is not yet fully understood. Microsecond freeze-hyperquenching single turnover experiments were carried out with fully reduced cytochrome aa(3) reacting with O(2) between 83 micros and 6 ms. Trapped intermediates were analyzed by low temperature UV-visible, X-band, and Q-band EPR spectroscopy, enabling determination of the oxidation-reduction kinetics of Cu(A), heme a, heme a(3), and of a recently detected tryptophan radical (Wiertz, F. G. M., Richter, O. M. H., Cherepanov, A. V., MacMillan, F., Ludwig, B., and de Vries, S. (2004) FEBS Lett. 575, 127-130). Cu(B) and heme a(3) were EPR silent during all stages of the reaction. Cu(A) and heme a are in electronic equilibrium acting as a redox pair. The reduction potential of Cu(A) is 4.5 mV lower than that of heme a. Both redox groups are oxidized in two phases with apparent half-lives of 57 micros and 1.2 ms together donating a single electron to the binuclear center in each phase. The formation of the heme a(3) oxoferryl species P(R) (maxima at 430 nm and 606 nm) was completed in approximately 130 micros, similar to the first oxidation phase of Cu(A) and heme a. The intermediate F (absorbance maximum at 571 nm) is formed from P(R) and decays to a hitherto undetected intermediate named F(W)(*). F(W)(*) harbors a tryptophan radical, identified by Q-band EPR spectroscopy as the tryptophan neutral radical of the strictly conserved Trp-272 (Trp-272(*)). The Trp-272(*) populates to 4-5% due to its relatively low rate of formation (t((1/2)) = 1.2 ms) and rapid rate of breakdown (t((1/2)) = 60 micros), which represents electron transfer from Cu(A)/heme a to Trp-272(*). The formation of the Trp-272(*) constitutes the major rate-determining step of the catalytic cycle. Our findings show that Trp-272 is a redox-active residue and is in this respect on an equal par to the metallocenters of the cytochrome c oxidase. Trp-272 is the direct reductant either to the heme a(3) oxoferryl species or to Cu (2+)(B). The potential role of Trp-272 in proton pumping is discussed.     

Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.     

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.     

Dioxygen reduction by bo-type quinol oxidase from Escherichia coli studied by submillisecond-resolved freeze-quench EPR spectroscopy

The mechanism of the dioxygen (O(2)) reduction conducted by cytochrome bo-type quinol oxidase was investigated using submillisecond-resolved freeze-quench EPR spectroscopy. The fully reduced form of the wild-type enzyme (WT) with the bound ubiquinone-8 at the high-affinity quinone-binding site was mixed with an O(2)-saturated solution, and the subsequent reaction was quenched at different time intervals from 0.2 to 50 ms. The EPR signals derived from the binuclear center and heme b were weak in the time domain from 0.2 to 0.5 ms. The signals derived from the ferric heme b and hydroxide-bound ferric heme o increased simultaneously after 1 ms, indicating that the oxidation of heme b is coupled to the formation of hydroxy heme o. In contrast, the enzyme without the bound ubiquinone-8 (Delta UbiA) showed the faster oxidation of heme b and the slower formation of hydroxy heme o than WT. It is interpreted that the F(I) intermediate possessing ferryl-oxo heme o, cupric Cu(B), and ferric heme b is converted to the F(II) intermediate within 0.2 ms by an electron transfer from the bound ubiquinonol-8 to ferric heme b. The conversion of the F(II) intermediate to the hydroxy intermediate occurred after 1 ms and was accompanied by the one-electron transfer from heme b to the binuclear center. Finally, it is suggested that the hydroxy intermediate possesses no bridging ligand between heme o and Cu(B) and is the final intermediate in the turnover cycle of cytochrome bo under steady-state conditions.     

Tryptophan-based radical in the catalytic mechanism of versatile peroxidase from Bjerkandera adusta

Versatile peroxidase (VP) from Bjerkandera adusta is a structural hybrid between lignin (LiP) and manganese (MnP) peroxidase. This hybrid combines the catalytic properties of the two above peroxidases, being able to oxidize typical LiP and MnP substrates. The catalytic mechanism is that of classical peroxidases, where the substrate oxidation is carried out by a two-electron multistep reaction at the expense of hydrogen peroxide. Elucidation of the structures of intermediates in this process is crucial for understanding the mechanism of substrate oxidation. In this work, the reaction of H(2)O(2) with the enzyme in the absence of substrate has been investigated with electron paramagnetic resonance (EPR) spectroscopy. The results reveal an EPR signal with partially resolved hyperfine structure typical of an organic radical. The yield of this radical is approximately 30%. Progressive microwave power saturation measurements indicate that the radical is weakly coupled to a paramagnetic metal ion, suggesting an amino acid radical in moderate distance from the ferryl heme. A tryptophan radical was identified as a protein-based radical formed during the catalytic mechanism of VP from Bjerkandera adusta through X-band and high-field EPR measurements at 94 GHz, aided by computer simulations for both frequency bands. A close analysis of the theoretical model of the VP from Bjerkandera sp. shows the presence of a tryptophan residue near to the heme prosthetic group, which is solvent-exposed as in the case of LiP and other VPs. The catalytic role of this residue in a long-range electron-transfer pathway is discussed.     

Formation of a pterin radical in the reaction of the heme domain of inducible nitric oxide synthase with oxygen

The heme domain (iNOS(heme)) of inducible nitric oxide synthase (NOS) was expressed in Escherichia coli and purified to homogeneity. Rapid freeze-quench (RFQ) EPR was used to monitor the reaction of the reduced iNOS(heme) with oxygen in the presence and absence of substrate. In these reactions, heme oxidation occurs at a rate of approximately 15 s(-)(1) at 4 degrees C. A transient species with a g = 2.0 EPR signal is also observed under these conditions. The spectral properties of the g = 2.0 signal are those of an anisotropic organic radical with S = (1)/(2). Comparison of the EPR spectra obtained when iNOS(heme) is reconstituted with N5-(14)N- and (15)N-substituted tetrahydrobiopterin (H(4)B) shows a hyperfine interaction with the pterin N5 nitrogen and identifies the radical as the one-electron oxidized form (H(3)B.) of the bound H(4)B. Substitution of D(2)O for H(2)O reveals the presence of hyperfine-coupled exchangeable protons in the H(4)B radical. This radical forms at a rate of 15-20 s(-)(1), with a slower decay rate that varies (0.12-0.7 s(-)(1)) depending on the substrate. At 127 ms, H(3)B. accumulates to a maximum of 80% of the total iNOS(heme) concentration in the presence of arginine but only to approximately 2.8% in the presence of NHA. Double-mixing RFQ experiments, where NHA is added after the formation of H(3)B., show that NHA does not react rapidly with H(3)B. and suggest that NHA instead prevents the formation of the H(4)B radical. These data constitute the first direct evidence for an NOS-bound H(3)B. and are most consistent with a role for H(4)B in electron transfer in the NOS reaction.     

Regulation of aldoxime dehydratase activity by redox-dependent change in the coordination structure of the aldoxime-heme complex

Phenylacetaldoxime dehydratase from Bacillus sp. strain OxB-1 (OxdB) catalyzes the dehydration of Z-phenylacetaldoxime (PAOx) to produce phenylacetonitrile. OxdB contains a protoheme that works as the active center of the dehydration reaction. The enzymatic activity of ferrous OxdB was 1150-fold higher than that of ferric OxdB, indicating that the ferrous heme was the active state in OxdB catalysis. Although ferric OxdB was inactive, the substrate was bound to the ferric heme iron. Electron paramagnetic resonance spectroscopy revealed that the oxygen atom of PAOx was bound to the ferric heme, whereas PAOx was bound to the ferrous heme in OxdB via the nitrogen atom of PAOx. These results show a novel mechanism by which the activity of a heme enzyme is regulated; that is, the oxidation state of the heme controls the coordination structure of a substrate-heme complex, which regulates enzymatic activity. Rapid scanning spectroscopy using stopped-flow apparatus revealed that a reaction intermediate (the PAOx-ferrous OxdB complex) showed Soret, alpha, and beta bands at 415, 555, and 524 nM, respectively. The formation of this intermediate complex was very fast, finishing within the dead time of the stopped-flow mixer (approximately 3 ms). Site-directed mutagenesis revealed that His-306 was the catalytic residue responsible for assisting the elimination of the hydrogen atom of PAOx. The pH dependence of OxdB activity suggested that another amino acid residue that assists the elimination of the OH group of PAOx would work as a catalytic residue along with His-306.     

Discovery of the true peroxy intermediate in the catalytic cycle of terminal oxidases by real-time measurement

The sequence of the catalytic intermediates in the reaction of cytochrome bd terminal oxidases from Escherichia coli and Azotobacter vinelandii with oxygen was monitored in real time by absorption spectroscopy and electrometry. The initial binding of O(2) to the fully reduced enzyme is followed by the fast (5 micros) conversion of the oxy complex to a novel, previously unresolved intermediate. In this transition, low spin heme b(558) remains reduced while high spin heme b(595) is oxidized with formation of a new heme d-oxygen species with an absorption maximum at 635 nm. Reduction of O(2) by two electrons is sufficient to produce (hydro)peroxide bound to ferric heme d. In this case, the O-O bond is left intact and the newly detected intermediate must be a peroxy complex of heme d (Fe (3+)(d)-O-O-(H)) corresponding to compound 0 in peroxidases. The alternative scenario where the O-O bond is broken as in the P(M) intermediate of heme-copper oxidases and compound I of peroxidases is not very likely, because it would require oxidation of a nearby amino acid residue or the porphyrin ring that is energetically unfavorable in the presence of the reduced heme b(558) in the proximity of the catalytic center. The formation of the peroxy intermediate is not coupled to membrane potential generation, indicating that hemes d and b(595) are located at the same depth of the membrane dielectric. The lifetime of the new intermediate is 47 micros; it decays into oxoferryl species due to oxidation of low spin heme b(558) that is linked to significant charge translocation across the membrane.     

Radical sites in Mycobacterium tuberculosis KatG identified using electron paramagnetic resonance spectroscopy, the three-dimensional crystal structure, and electron transfer couplings

Catalase-peroxidase (KatG) from Mycobacterium tuberculosis, a Class I peroxidase, exhibits high catalase activity and peroxidase activity with various substrates and is responsible for activation of the commonly used antitubercular drug, isoniazid (INH). KatG readily forms amino acid-based radicals during turnover with alkyl peroxides, and this work focuses on extending the identification and characterization of radicals forming on the millisecond to second time scale. Rapid freeze-quench electron paramagnetic resonance spectroscopy (RFQ-EPR) reveals a change in the structure of the initially formed radical in the presence of INH. Heme pocket binding of the drug and knowledge that KatG[Y229F] lacks this signal provides evidence for radical formation on residue Tyr(229). High field RFQ-EPR spectroscopy confirmed a tryptophanyl radical signal, and new analyses of X-band RFQ-EPR spectra also established its presence. High field EPR spectroscopy also confirmed that the majority radical species is a tyrosyl radical. Site-directed mutagenesis, along with simulations of EPR spectra based on x-ray structural data for particular tyrosine and tryptophan residues, enabled assignments based on predicted hyperfine coupling parameters. KatG mutants W107F, Y229F, and the double mutant W107F/Y229F showed alteration in type and yield of radical species. Results are consistent with formation of a tyrosyl radical reasonably assigned to residue Tyr(229) within the first few milliseconds of turnover. This is followed by a mixture of tyrosyl and tryptophanyl radical species and finally to only a tyrosyl radical on residue Tyr(353), which lies more distant from the heme. The radical processing of enzyme lacking the Trp(107)-Tyr(229)-Met(255) adduct (found as a unique structural feature of catalase-peroxidases) is suggested to be a reasonable assignment of the phenomena.     

Identification and characterization of tyrosyl radical formation in Mycobacterium tuberculosis catalase-peroxidase (KatG)

The catalytic function of Mycobacterium tuberculosis catalase-peroxidase (KatG) and its role in activation of the anti-tuberculosis antibiotic isoniazid were investigated using rapid freeze-quench electron paramagnetic resonance (RFQ-EPR) experiments. The reaction of KatG with peroxyacetic acid was followed as a function of time using x-band EPR at 77 K. A doublet EPR signal appears within 6.4 ms after mixing and at time points through hundreds of milliseconds. Thereafter, a singlet signal develops and finally predominates after 1 s, with a total yield of radical approximately 0.5 spin/heme. Simulation of the spectra provided EPR parameters consistent with those for tyrosyl radicals. Changes in the hyperfine splitting and/or line width in spectra for l-3,3-[2H2]tyrosine-labeled, but not l-2,4,5,6,7-[2H5]tryptophan-labeled KatG confirmed this assignment. The initial rate of radical formation was unchanged using a 3-fold or 10-fold excess of peroxyacetic acid, consistent with a rate-determining step involving an intermediate. Although Compound I is likely to be the precursor of tyrosyl radical in KatG, neither its EPR signal nor its reduction to Compound II during formation of the radical(s) could be observed. The tyrosyl radical doublet signal was rapidly quenched by addition of isoniazid and benzoic hydrazide, but not by iproniazid, which binds poorly to KatG.     

Water-hydroxide exchange reactions at the catalytic site of heme-copper oxidases

Membrane-bound heme-copper oxidases catalyze the reduction of O(2) to water. Part of the free energy associated with this process is used to pump protons across the membrane. The O(2) reduction reaction results in formation of high-pK(a) protonatable groups at the catalytic site. The free energy associated with protonation of these groups is used for proton pumping. One of these protonatable groups is OH(-), coordinated to the heme and Cu(B) at the catalytic site. Here we present results from EPR experiments on the Rhodobacter sphaeroides cytochrome c oxidase, which show that at high pH (9) approximately 50% of oxidized heme a(3) is hydroxide-ligated, while at low pH (6.5), no hydroxide is bound to heme a(3). The kinetics of hydroxide binding to heme a(3) were investigated after dissociation of CO from heme a(3) in the enzyme in which the heme a(3)-Cu(B) center was reduced while the remaining redox sites were oxidized. The dissociation of CO results in a decrease of the midpoint potential of heme a(3), which results in electron transfer (tau approximately equal 3 micros) from heme a(3) to heme a in approximately 100% of the enzyme population. At pH >7.5, the electron transfer is followed by proton release from a H(2)O molecule to the bulk solution (tau approximately equal 2 ms at pH 9). This reaction is also associated with absorbance changes of heme a(3), which on the basis of the results from the EPR experiments are attributed to formation of hydroxide-ligated heme a(3). The OH(-) bound to heme a(3) under equilibrium conditions at high pH is also formed transiently after O(2) reduction at low pH. It is proposed that the free energy associated with electron transfer to the binuclear center and protonation of this OH(-) upon reduction of the recently oxidized enzyme provides the driving force for the pumping of one proton.     

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.     

Spectroscopic studies of peroxyacetic acid reaction intermediates of cytochrome P450cam and chloroperoxidase

It is generally assumed that the putative compound I (cpd I) in cytochrome P450 should contain the same electron and spin distribution as is observed for cpd I of peroxidases and catalases and many synthetic cpd I analogues. In these systems one oxidation equivalent resides on the Fe(IV)=O unit (d(4), S=1) and one is located on the porphyrin (S'=1/2), constituting a magnetically coupled ferryl iron-oxo porphyrin pi-cation radical system. However, this laboratory has recently reported detection of a ferryl iron (S=1) and a tyrosyl radical (S'=1/2), via M?ssbauer and EPR studies of 8 ms-reaction intermediates of substrate-free P450cam from Pseudomonas putida, prepared by a freeze-quench method using peroxyacetic acid as the oxidizing agent [Schünemann et al., FEBS Lett. 479 (2000) 149]. In the present study we show that under the same reaction conditions, but in the presence of the substrate camphor, only trace amounts of the tyrosine radical are formed and no Fe(IV) is detectable. We conclude that camphor restricts the access of the heme pocket by peroxyacetic acid. This conclusion is supported by the additional finding that binding of camphor and metyrapone inhibit heme bleaching at room temperature and longer reaction times, forming only trace amounts of 5-hydroxy-camphor, the hydroxylation product of camphor, during peroxyacetic acid oxidation. As a control we performed freeze-quench experiments with chloroperoxidase from Caldariomyces fumago using peroxyacetic acid under the identical conditions used for the substrate-free P450cam oxidations. We were able to confirm earlier findings [Rutter et al., Biochemistry 23 (1984) 6809], that an antiferromagnetically coupled Fe(IV)=O porphyrin pi-cation radical system is formed. We conclude that CPO and P450 behave differently when reacting with peracids during an 8-ms reaction time. In P450cam the formation of Fe(IV) is accompanied by the formation of a tyrosine radical, whereas in CPO Fe(IV) formation is accompanied by the formation of a porphyrin radical.     

Detection of reduced RNA synthesis in UV-irradiated Cockayne syndrome group B cells using an isolated nuclear system

In aqueous methyl linoleate emulsions (pH 7.4, 25 degrees C, air-saturated), nitrosylmyoglobin and saturated fatty acid anions (palmitate and stearate investigated) each showed antioxidant effect on metmyoglobin-induced peroxidation as measured by oxygen depletion rate. For equimolar concentration of nitrosylmyoglobin and metmyoglobin and for metmyoglobin in moderate excess, a reduction in oxygen consumption rate of approximately 70% was observed. Fatty acid anions reduced oxygen consumption rate most significantly for palmitate (up to 60% for a fatty acid:heme protein ratio of 90:1). No further antioxidative effect was seen for fatty acid anions in the presence of nitrosylmyoglobin, whereas nitrosylmyoglobin showed a further antioxidant effect in presence of fatty acid anions in the metmyoglobin-catalyzed process. The antioxidative mechanism of nitrosylmyoglobin and fatty acid anions is different, and while the fatty acid anions seem active in inhibiting initiation of oxidation through protection against metmyoglobin activation into perferrylmyoglobin, as shown by freeze-quench Electron Spin Resonance (ESR) spectroscopy, nitrosylmyoglobin is rather active in the oxygen consuming (propagation) phase.     

Design of turbulent tangential micro-mixers that mix liquids on the nanosecond time scale

Unravelling (bio)chemical reaction mechanisms and macromolecular folding pathways on the (sub)microsecond time scale is limited by the time resolution of kinetic instruments for mixing reactants and observation of the progress of the reaction. To improve the mixing time resolution, turbulent four- and two-jet tangential micro-mixers were designed and characterized for their mixing and (unwanted) premixing performances employing acid–base reactions monitored by a pH-sensitive fluorescent dye. The mixing performances of the micro-mixers were determined after the mixing chamber in a free-flowing jet. The premixing behavior in the vortex chamber was assessed in an optically transparent glass–silicon replica of a previously well-characterized stainless-steel four-jet tangential micro-mixer. At the highest flow rates, complete mixing was achieved in 160 ns with only approximately 9% premixing of the reactants. The mixing time of 160 ns is at least 50 times shorter than estimated for other fast mixing devices. Key aspects to the design of ultrafast turbulent micro-mixers are discussed. The integration of these micro-mixers with an optical flow cell would enable the study of the very onset of chemical reactions in general and of enzyme catalytic reactions in particular.     

Spectroscopic and substrate binding properties of heme-containing aldoxime dehydratases, OxdB and OxdRE

Aldoxime dehydratase (Oxd) is a novel hemeprotein that catalyzes the dehydration reaction of aldoxime to produce nitrile. In this study, we studied the spectroscopic and substrate binding properties of two Oxds, OxdB from Bacillus sp. strain OxB-1 and OxdRE from Rhodococcus sp. N-771, that show different quaternary structures and relatively low amino acid sequence identity. Electronic absorption and resonance Raman spectroscopy revealed that ferric OxdRE contained a six-coordinate low-spin heme, while ferric OxdB contained a six-coordinate high-spin heme. Both ferrous OxdRE and OxdB included a five-coordinate high-spin heme to which the substrate was bound via its nitrogen atom for the reaction to occur. Although the ferric Oxds were inactive for catalysis, the substrate was bound to the ferric heme via its oxygen atom in both OxdB and OxdRE. Electronic paramagnetic resonance (EPR) and rapid scanning spectroscopy revealed that the flexibility of the heme pocket was different between OxdB and OxdRE, which might affect their substrate specificity.     

A continuous-flow capillary mixing method to monitor reactions on the microsecond time scale.

A continuous-flow capillary mixing apparatus, based on the original design of Regenfuss et al. (Regenfuss, P., R. M. Clegg, M. J. Fulwyler, F. J. Barrantes, and T. M. Jovin. 1985. Rev. Sci. Instrum. 56:283-290), has been developed with significant advances in mixer design, detection method and data analysis. To overcome the problems associated with the free-flowing jet used for observation in the original design (instability, optical artifacts due to scattering, poor definition of the geometry), the solution emerging from the capillary is injected directly into a flow-cell joined to the tip of the outer capillary via a ground-glass joint. The reaction kinetics are followed by measuring fluorescence versus distance downstream from the mixer, using an Hg(Xe) arc lamp for excitation and a digital camera with a UV-sensitized CCD detector for detection. Test reactions involving fluorescent dyes indicate that mixing is completed within 15 micros of its initiation and that the dead time of the measurement is 45 +/- 5 micros, which represents a >30-fold improvement in time resolution over conventional stopped-flow instruments. The high sensitivity and linearity of the CCD camera have been instrumental in obtaining artifact-free kinetic data over the time window from approximately 45 micros to a few milliseconds with signal-to-noise levels comparable to those of conventional methods. The scope of the method is discussed and illustrated with an example of a protein folding reaction.     

Two sub-states of the red2 state of methyl-coenzyme M reductase revealed by high-field EPR spectroscopy

Methyl-coenzyme M reductase (MCR) catalyzes the formation of methane from methyl-coenzyme M and coenzyme B in methanogenic archaea. The enzyme has two structurally interlinked active sites embedded in an α₂β₂γ₂ subunit structure. Each active site has the nickel porphyrinoid F₄₃₀ as a prosthetic group. In the active state, F₄₃₀ contains the transition metal in the Ni(I) oxidation state. The active enzyme exhibits an axial Ni(I)-based continuous wave (CW) electron paramagnetic resonance (EPR) signal, called red1a in the absence of substrates or red1c in the presence of coenzyme M. Addition of coenzyme B to the MCR-red1 state can partially and reversibly convert it into the MCR-red2 form, which shows a rhombic Ni(I)-based EPR signal (at X-band microwave frequencies of approximately 9.4 GHz). In this report we present evidence from high-field/high-frequency CW EPR spectroscopy (W-band, microwave frequency of approximately 94 GHz) that the red2 state consists of two substates that could not be resolved by EPR spectroscopy at X-band frequencies. At W-band it becomes apparent that upon addition of coenzyme B to MCR in the red1c state, two red2 EPR signals are induced, not one as was previously believed. The first signal is the well-characterized (ortho)rhombic EPR signal, thus far called red2, while the second previously unidentified signal is axial. We have named the two substates MCR-red2r and MCR-red2a after their rhombic and axial signals, respectively. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.