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 molybd o-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherich ia coli nitrate reductase (NarGHI). Preparation of fluorescent oxidize d derivatives of MGD (Form A and Form B) indicates that in a mobAB mut ant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzym e. Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistr y with respect to the dithionite reducibility of the [Fe-S] clusters o f NarH. Potentiometric titrations of membrane-bound NarGHI indicate th at the NarH [Fe-S] clusters have midpoint potentials at pH 8.0 (E-m,E- 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 -4 20 mV in a mobAB mutant background. These data support the following c onclusions: (i) a model for Mo-MGD biosynthesis and assembly into NarG HI in which both metal chelation and nucleotide addition to molybdopte rin precede cofactor insertion; and (ii) the absence of Mo-MGD signifi cantly affects E-m,E- 8.0, of the highest potential [4Fe4S]cluster.