Fourier‐transform infrared studies on azide‐binding to the binuclear center of the <i>Escherichia coli bo</i>‐type ubiquinol oxidase<sup>1</sup>

Tsubaki, Motonari; Mogi, Tatsushi; Hori, Hiroshi

doi:10.1016/s0014-5793(99)00423-8

Cited by 11 publications

(6 citation statements)

References 27 publications

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“…5(d)] in the active site pocket to compensate for the inequivalence of the two N-N bonds of the bound azide (44,45). Indeed the pattern of the azide isotopic shifts observed in the present study was very different from those of heme-copper oxidases in the azide-bridging configuration (22,46), indicating an intrinsic difference in the azidebinding.…”

Section: Discussionmentioning

confidence: 54%

Azide- and Cyanide-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies

Tsubaki¹,

Mogi²,

Hori³

1999

Journal of Biochemistry

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Cytochrome bd-type ubiquinol oxidase contains two hemes b (b(558) and b(595)) and one heme d as the redox metal centers. To clarify the structure of the reaction center, we analyzed Escherichia coli cytochrome bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide ((14)N(3)) anti-symmetric stretching infrared band (2, 010.5 cm(-1)) showed anomalies upon (15)N-substitutions, indicating interactions with surrounding protein residues or heme b(595) in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at g = 3.19 and g = 7.15, which could arise from the heme d(Fe(3+))-CN-heme b(595)(Fe(3+)) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the g(z) = 2.85 signal ascribed to the minor heme b(595)-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme b(595).

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Section: Discussionmentioning

confidence: 54%

Azide- and Cyanide-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies

Tsubaki¹,

Mogi²,

Hori³

1999

Journal of Biochemistry

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“…An absorption at 2043 cm –1 , which is downshifted from the free azide ion (2049 cm –1 ), is observed within the swArM (Figure S11 and Table S3). This downshift is similar in frequency to that observed for azide ligated to the metal sites in high-spin heme of myoglobin and hemoglobin, Cu–Zn superoxide dismutase, cytochrome c oxidase, and other enzymes. − Upon Gln binding, we observe that the frequency again downshifts, now by ∼1 to 2042 cm –1 , with an additional small shoulder appearing at lower frequency (Figure S11). These observations demonstrate our ability to modulate the microenvironment of the metallocofactor via protein conformation.…”

Section: Resultsmentioning

confidence: 99%

Engineering a Conformationally Switchable Artificial Metalloprotein

et al. 2022

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Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3 –, H3C–, and i Pr–). We used UV–vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co–S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co–S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

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“…− with the release of H + . Both cyanide and azide can bridge between the ferric heme o 3 and cupric Cu B forming the following structures: Fe o3 3+ -C=N-Cu B 2+ and Fe o3 3+ -N=N=N-Cu B 2+ , where Fe o3 is the heme o 3 iron [55,57,58]. Compared to these two ligands, ammonia is a much smaller molecule.…”

Section: Proposed Mechanism For Inhibition Of Cytochrome Bo 3 By Ammoniamentioning

confidence: 99%

In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

2020

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Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

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Fourier‐transform infrared studies on azide‐binding to the binuclear center of the Escherichia coli bo‐type ubiquinol oxidase¹

Cited by 11 publications

References 27 publications

Azide- and Cyanide-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies

Azide- and Cyanide-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies

Engineering a Conformationally Switchable Artificial Metalloprotein

In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

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Fourier‐transform infrared studies on azide‐binding to the binuclear center of the Escherichia coli bo‐type ubiquinol oxidase1

Cited by 11 publications

References 27 publications

Azide- and Cyanide-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies

Azide- and Cyanide-Binding to the Escherichia coli bd-Type Ubiquinol Oxidase Studied by Visible Absorption, EPR and FTIR Spectroscopies

Engineering a Conformationally Switchable Artificial Metalloprotein

In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

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Fourier‐transform infrared studies on azide‐binding to the binuclear center of the Escherichia coli bo‐type ubiquinol oxidase¹