Electron Transfer from HemebLto the [3Fe-4S] Cluster ofEscherichia coliNitrate Reductase A (NarGHI)

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Abstract
We have investigated the functional relationship between three of the prosthetic groups of Escherichia coli nitrate reductase A (NarGHI): the two hemes of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster of the electron-transfer subunit (NarH). In two site-directed mutants (NarGHIH56R and NarGHIH205Y) that lack the highest potential heme of NarI (heme bH), a large negative ΔEm,7 is elicited on the NarH [3Fe-4S] cluster, suggesting a close juxtaposition of these two centers in the holoenzyme. In a mutant retaining heme bH, but lacking heme bL (NarGHIH66Y), there is no effect on the NarH [3Fe-4S] cluster redox properties. These results suggest a role for heme bH in electron transfer to the [3Fe-4S] cluster. Studies of the pH dependence of the [3Fe-4S] cluster, heme bH, and heme bL Em values suggest that significant deprotonation is only observed during oxidation of the latter heme (a pH dependence of −36 mV pH-1). In NarI expressed in the absence of NarGH [NarI(ΔGH)], apparent exposure of heme bH to the aqueous milieu results in both it and heme bL having Em values with pH dependencies of approximately −30 mV pH-1. These results are consistent with heme bH being isolated from the aqueous milieu and pH effects in the holoenzyme. Optical spectroscopy indicates that inhibitors such as HOQNO and stigmatellin bind and inhibit oxidation of heme bL but do not inhibit oxidation of heme bH. Fluorescence quench titrations indicate that HOQNO binds with higher affinity to the reduced form of NarGHI than to the oxidized form. Overall, the data support the following model for electron transfer through the NarI region of NarGHI: QP site → heme bL → heme bH → [3Fe-4S] cluster.