Rate‐Contributing Proton Transfers And Crystal Structures Of The Flavoenzyme Nitroreductase

The nitroreductase of Enterobacter cloacae typifies numerous related enzymes that reduce nitroaromatics to corresponding nitroso and hydroxylamino aromatics via sequential two‐electron reductions. To elucidate the sources of accompanying protons the primary and solvent kinetic isotope effects (KIEs) have been analyzed revealing that a robust primary KIE of 3.2 ± 0.1 applies to flavin reduction by NADH, suggesting hydride transfer. For the oxidative half reaction a primary KIE of 1.6 ± 0.1 indicates hydride transfer to para ‐nitrobenzoic acid ( p ‐NBA) from the flavin, subject to exchange of the N(5)H with solvent. Solvent KIEs are consistent with solvent as the source of the second proton in a synchronous (coupled) mechanism. Two new crystal structures have been solved, modeling the enzyme‐substrate complexes of the two half‐reactions. Nicotinic acid adenine dinucleotide serves as an analog for NADH and binds in an extended conformation with the nicotinic acid stacked on the re face of the flavin and the C(4) position only 3 Å from the flavin N(5), consistent with hydride transfer. P ‐NBA also stacks on the flavin's re face but the crystallographic data are not conclusive as to whether the carboxyl or nitro group is closer to the flavin N(5). The finding of hydride transfer supports binding of p ‐NBA with its nitro group nearer to N(5), in the reduced state of NR at least. Use of water as the second H donor concurs with nitroreductase's broad substrate repertoire. Support or Funding Information With support from the University of Kentuckty College of Arts and Sciences and the Center for Pharmaceutical Development, NSF IUCRC.Cartoon of active site of nitroreductase with substrate p‐NBA bound in one of the two crystallographically‐acceptable conformations. Flavin in yellow, paranitrobenzoic acid in purple, protein contributions in pale blue, all with heteroatoms in CPK colours.