Ligand interactions at the active site of aspartate transcarbamoylase from Escherichia coli

NO and O2 are detoxified in many organisms using flavodiiron proteins (FDPs). The exact coordination of the iron centre in the active site of these enzymes remains unclear despite numerous structural studies. Here, we used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand interactions in Escherichia coli FDP. This data combined with density functional theory (DFT) and 57Fe Mössbauer spectroscopy indicate that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)–(µOH–)2–Fe(III) active site, while its reduction gives rise to a monohydroxo-diferrous Fe(II)–(µOH–)–Fe(II) site upon elimination of one bridging OH– ligand, thereby providing an open coordination site for NO binding. Prolonged NRVS data collection of the oxidised FDP resulted in photoreduction and formation of a partially reduced diiron center with two bridging hydroxyl ligands. These results have crucial implications for studying and understanding the mechanism of FDP as well as other non-haem diiron enzymes.

Download Full-text

Arginine 54 in the active site of escherichia coli aspartate transcarbamoylase is critical for catalysis: A site-specific mutagenesis, NMR, and X-ray crystallographic study

Protein Science ◽

10.1002/pro.5560011105 ◽

1992 ◽

Vol 1 (11) ◽

pp. 1435-1446 ◽

Cited By ~ 18

Author(s):

Jeffrey W. Stebbins ◽

Diane E. Robertson ◽

Mary F. Roberts ◽

Raymond C. Stevens ◽

William N. Lipscomb ◽

...

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Aspartate Transcarbamoylase ◽

Site Specific ◽

X Ray ◽

Crystallographic Study ◽

A Site

Download Full-text

Glutamic acid 86 is important for positioning the 80's loop and arginine 54 at the active site of Escherichia coli aspartate transcarbamoylase and for the structural stabilization of the C1-C2 interface.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)31435-7 ◽

1994 ◽

Vol 269 (40) ◽

pp. 24608-24614

Author(s):

D.P. Baker ◽

J.W. Stebbins ◽

E. DeSena ◽

E.R. Kantrowitz

Keyword(s):

Escherichia Coli ◽

Glutamic Acid ◽

Active Site ◽

Aspartate Transcarbamoylase ◽

Structural Stabilization

Download Full-text

Hydroxo-Bridged Active Site of Flavodiiron NO Reductase Revealed by Spectroscopy and Computations

10.26434/chemrxiv.12928331 ◽

2020 ◽

Author(s):

Filipe Folgosa ◽

Vladimir Pelmenschikov ◽

Matthias Keck ◽

Christian Lorent ◽

Yoshitaka Yoda ◽

...

Keyword(s):

Escherichia Coli ◽

Density Functional Theory ◽

Data Collection ◽

Active Site ◽

Density Functional ◽

Nuclear Resonance ◽

Structural Studies ◽

Functional Theory ◽

Nuclear Resonance Vibrational Spectroscopy ◽

Ligand Interactions

NO and O2 are detoxified in many organisms using flavodiiron proteins (FDPs). The exact coordination of the iron centre in the active site of these enzymes remains unclear despite numerous structural studies. Here, we used 57Fe nuclear resonance vibrational spectroscopy (NRVS) to probe the iron-ligand interactions in Escherichia coli FDP. This data combined with density functional theory (DFT) and 57Fe Mössbauer spectroscopy indicate that the oxidised form of FDP contains a dihydroxo-diferric Fe(III)–(µOH–)2–Fe(III) active site, while its reduction gives rise to a monohydroxo-diferrous Fe(II)–(µOH–)–Fe(II) site upon elimination of one bridging OH– ligand, thereby providing an open coordination site for NO binding. Prolonged NRVS data collection of the oxidised FDP resulted in photoreduction and formation of a partially reduced diiron center with two bridging hydroxyl ligands. These results have crucial implications for studying and understanding the mechanism of FDP as well as other non-haem diiron enzymes.

Download Full-text