PMID:9516445
| Citation |
Rothery, RA, Magalon, A, Giordano, G, Guigliarelli, B, Blasco, F and Weiner, JH (1998) The molybdenum cofactor of Escherichia coli nitrate reductase A (NarGHI). Effect of a mobAB mutation and interactions with [Fe-S] clusters. J. Biol. Chem. 273:7462-9 |
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| Abstract |
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. |
| Links | |
| Keywords |
Binding Sites/genetics; Coenzymes/metabolism; Electron Spin Resonance Spectroscopy; Escherichia coli/enzymology; Guanine Nucleotides/genetics; Guanine Nucleotides/metabolism; Hydrogen-Ion Concentration; Iron-Sulfur Proteins/metabolism; Molybdenum/metabolism; Mutagenesis, Site-Directed; Nitrate Reductase; Nitrate Reductases/genetics; Nitrate Reductases/metabolism; Organometallic Compounds/metabolism; Spectrometry, Fluorescence; Tungsten/pharmacology |
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