The molybdenum cofactor of Escherichia coli nitrate reductase A (NarGHI). Effect of a mobAB mutation and interactions with [Fe-S] clusters.
We have studied the effect of a mobAB mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherichia coli nitrate reductase (NarGHI). Preparation of fluorescent oxidized derivatives of MGD (Form A and Form B) indicates that in a mobAB mutant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzyme. Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistry with respect to the dithionite reducibility of the [Fe-S] clusters of NarH. Potentiometric titrations of membrane-bound NarGHI indicate that the NarH [Fe-S] clusters have midpoint potentials at pH 8.0 (Em,8.0 values) of +180 mV ([3Fe-4S] cluster), +130, -55, and -420 mV ([4Fe-4S] clusters) in a wild-type background and +180, +80, -35, and -420 mV in a mobAB mutant background. These data support the following conclusions: (i) a model for Mo-MGD biosynthesis and assembly into NarGHI in which both metal chelation and nucleotide addition to molybdopterin precede cofactor insertion; and (ii) the absence of Mo-MGD significantly affects Em,8.0 of the highest potential [4Fe-4S] cluster.
Rothery
R A
RA
Medical Research Council Group in the Molecular Biology of Membranes, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Magalon
A
A
Giordano
G
G
Guigliarelli
B
B
Blasco
F
F
Weiner
J H
JH
D001665:Binding Sites / Q000235:genetics; D003067:Coenzymes / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008982:Molybdenum / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics / Q000378:metabolism*; D009942:Organometallic Compounds / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014414:Tungsten / Q000494:pharmacology
D003067:Coenzymes; D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D009942:Organometallic Compounds; C106961:bis(molybdopterin guanine dinucleotide)molybdenum; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D014414:Tungsten