The molybdenum cofactor of Escherichia coli nitrate reductase a (NarGHI): Effect of a mobAB mutation and interactions with [Fe-S] clusters

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) fores 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 (E(m,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 E(m,8.0) of the highest potential [4Fe4S] cluster.