Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

Suzuki, Shinnichiro; Deligeer,; Yamaguchi, Kazuya; Kataoka, Keisuke; Kobayashi, Kazuo; Tagawa, Seiichi; Kohzuma, Takamitsu; Shidara, Sohsuke; Iwasaki, Hidekazu

doi:10.1007/s007750050132

Cited by 109 publications

(133 citation statements)

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“…In agreement with the earlier observations of Suzuki et al for the reactivity of AcNiR, AxgNiR and AxnNiR [12,13], a direct bimolecular reduction of the T1 Cu(II) site by NMNA 3 radicals was observed with a second order rate constant of k PWV = (5 þ 1)U10 V M 3I s 3I . We found this rate to be the same, within experimental error, when gy 3 2 radicals were used as the electron donor.…”

Section: Resultssupporting

confidence: 92%

“…1, the amplitude of the increase in absorbance associated with the reoxidation of the T1 Cu(I) centre during the intramolecular ET from T1 Cu(I) to T2 Cu(II) does not correspond to the complete reoxidation of the T1 site. A similar observation was made in the earlier pulse radiolysis studies [7,12,13]. Since the midpoint potential of T1 Cu(II)/Cu(I) for AxNiR has been reported to be 240 mV at 298 K [12] one may calculate a midpoint potential of T2 Cu(II)/Cu(I) from the above ET equilibrium to be 230 mV.…”

Section: Resultssupporting

confidence: 72%

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The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

et al. 1998

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The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis. The reversible ET reaction proceeds with a rate constant k i = k I3P +k P3I of 450 þ 30 s 3I at pH 7.0 and 298 K. The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k I3P = 185 þ 12 s 3I and k P3I 265 þ 18 s 3I . The temperature dependence of K allowed us to determine the v vH³ value of the ET equilibrium to be 12.1 kJ mol 3I . Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, v vH g = 22.7 þ 3.4 kJ mol 3I and v vS g = 3126 þ 11 J K 3I mol 3I ; backward reaction, v vH g = 10.6 þ 1.7 kJ mol 3I and v vS g = 3164 þ 15 J K 3I mol 3I . X-ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys IQT , which provides the thiolate ligand to the type 1 copper ion, and the adjacent His IQS residue with its imidazole being one of the ligands to the type 2 Cu ion. This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase.z 1998 Federation of European Biochemical Societies.

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Section: Resultssupporting

confidence: 92%

Section: Resultssupporting

confidence: 72%

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

et al. 1998

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“…However, they refer to experiments in the absence of nitrite. Suzuki et al (29) have reported that these rates drop, in some cases by Ͼ1 order of magnitude, in the presence of nitrite (but still in the absence of turnover). It is generally accepted now that binding and turnover of nitrite may cause subtle structural changes in the protein framework around the catalytic site that may considerably affect the rate of intramolecular electron transfer.…”

Section: Discussionmentioning

confidence: 99%

The enzyme mechanism of nitrite reductase studied at single-molecule level

Кузнецова

Zauner

Aartsma

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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A generic method is described for the fluorescence ''readout'' of the activity of single redox enzyme molecules based on Fö rster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to singlemolecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.electron transfer ͉ redox enzyme ͉ Fö rster transfer ͉ nitric oxide ͉ fluorescent label I n the past few years, single-enzyme studies have revealed numerous hidden aspects of enzyme behavior (1). The huge potential of these studies to unravel the intricate kinetics and precise workings of enzymes, which are often hidden within the ensemble properties, is somewhat restricted by current approaches. The majority of existing single-molecule enzymatic assays are based on fluorescence and have been limited to the flavoenzymes, which contain a fluorescent cofactor (2-5), or to enzymes for which a suitable fluorogenic substrate could be designed (6-9). Recently, it was shown how redox enzyme activity in the bulk can be studied by Förster resonance energy transfer (FRET) from an attached fluorescent label to the enzyme cofactor (10, 11). Here, we report, first, how this technique can be successfully applied to study the enzymatic turnover of single surface-confined copper-containing nitrite reductase (NiR) molecules labeled with ATTO 655 using scanning confocal fluorescence microscopy. Second, it is shown how the kinetics of the fluorescence time traces can be connected with the kinetic parameters that describe the ensemble behavior of the enzyme. Finally, the rate of the electron transfer between the types 1 and 2 Cu centers during turnover could be established. The observed distribution of rates is connected to the partial structural disorder in the catalytic site that has been observed in crystallographic studies. ResultsEnzyme Mechanism. Dissimilatory copper-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6 converts nitrite into nitric oxide. The enzyme is a homotrimer, each monomer containing one type 1 and one type 2 copper site (Fig. 1). The type 1 copper accepts an electron from the physiological donor and transfers it to the type 2 copper, where nitrite is reduced to nitric oxide (NO). The midpoint potentials of the types 1 and 2 Cu centers are close, resulting in a redox equilibrium constant for the two sites that is close to 1 (12, 13). Regarding the enzyme mechanism, it has been stated (14, 15) that, after reduction of the type 1 Cu site, the electron is passed on to the type 2...

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“…Electron-nuclear double resonance measurements (30) and crystallographic studies (31) on the T2 Cu suggest a greater thermodynamic driving force for ET after nitrite bonding due to weaker coordination of His ligands, and a consequent increase in rate constant. In contrast, pulse radiolysis measurements showed a sharp decrease in rate upon nitrite binding (16). Bulk activity assays and electrochemical measurements have suggested that an increase in ET rate constant upon nitrite binding is expected at higher pH values, whereas a decrease upon binding is expected at lower pH, depending on whether the nitrite displaces a water molecule or hydroxide ion (21).…”

Section: Discussionmentioning

confidence: 99%

“…Intramolecular electron transfer (ET) (14) over 12.6 Å results in reduction of the proximal type 2 (T2) Cu, which is located at the monomer-monomer interface and is coordinated by three His residues (13). One of these T2 Cu ligands is adjacent to the Cys residue at the T1 site, and this covalent network has been proposed as the pathway for ET (15)(16)(17). Nitrite reduction occurs at the T2 Cu with protons for water formation being provided by a hydrogen bond network composed of a bound water, and the second coordination sphere Asp92 and His249 residues (18).…”

mentioning

confidence: 99%

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

Goldsmith

Tabares

Kostrz

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.ABEL trap | enzymology | electron transfer | fluorescence | catalysis M easurements made on the activity of single enzymes have revolutionized the picture of these molecular machines' mode of operation by providing evidence for static and dynamic heterogeneity, enabling the observation of transient intermediates, and allowing determination of the microscopic rate constants comprising complex catalytic cycles under turnover conditions (1-4). However, conclusions regarding enzyme dynamics require the observation of multiple turnovers of a single molecule with a requisite observation window of several seconds or more. Because this window is several orders of magnitude longer than the typical dwell time of a solution-phase molecule diffusing through a confocal detection volume, molecules that naturally exist in solution are often immobilized on a solid support (1-3, 5). Although gross comparison between properties measured on immobilized and freely diffusing biomolecules tends to show good agreement (1, 3), more subtle aspects of biomolecule behavior such as property distribution widths and shapes (6, 7) and conformational dynamics (6, 8) have been shown to be affected by immobilization, and in some cases, immobilization has been suspected of contributing to observed enzyme behavioral heterogeneity (4). In this report we take an important step toward viewing the richness of single-enzyme dynamics without immobilization by making observations of multiple turnover events of single solution-phase molecules of a blue nitrite reductase (bNiR), an important redox-active enzyme in bacterial denitrification (9).Prolonged solution-phase measurements are enabled through use of a specialized microfluidic trapping device that cancels the Brownian motion of a single emissive solution-phase object through the use of directed electroosmotic forces, the anti-Brownian electrokinetic (ABEL) trap (10, 11). The ABEL trap estimates particle position via a revolving laser spot phase-locked in a homebuilt analogue circuit to a restoring force gated by photon detection and applied via two orthogonal pairs of electrodes (11). This scheme results in the confinement of the object in a spatially homogeneous excitation volume for multiple seconds without immobilization and can be applied to single biomolecules substantially smaller than the large...

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Spectroscopic characterization and intramolecular electron transfer processes of native and type 2 Cu-depleted nitrite reductases

Cited by 109 publications

References 0 publications

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase

The enzyme mechanism of nitrite reductase studied at single-molecule level

Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

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