Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

The structure of the Escherichia coli flavodoxin NADP(+) oxidoreductase (FL DR) places three arginines (R144, R174 and R184) in the proposed NADPH-bind ing site. Mutant enzymes produced by site-directed mutagenesis, in which ea ch arginine was replaced by neutral alanine, were characterized. All mutant s exhibited decreased NADPH-dependent cytochrome c reductase activity (R144 A, 241.6 min(-1); R174A, 132.1 min(-1); R184A, 305.5 min(-1) versus wild ty pe, 338.9 min(-1)) and increased K-m for NADPH (R144A, 5.3 muM; R174A, 20.2 muM; R184A, 54.4 muM versus wild type, 3.9 muM). The k(cat) value for NADH -dependent cytochrome c reduction was increased for R174A (42.3 min(-1)) an d R184A (50.4 min(-1)) compared with the wild type (33.0 min(-1)), consiste nt with roles for R174 and R184 in discriminating between NADPH/NADH by int eraction with the adenosine ribose 2'-phosphate. Stopped-flow studies indic ated that affinity (K-d) for NADPH was markedly reduced in mutants R144A (6 35 muM) and R184A (2.3 mM) compared with the wild type (< 5 <mu>M). Mutant R184A displays the greatest change in pyridine nucleotide preference, with the NADH/NADPH K-d ratio > 175-fold lower than for wild-type FLDR. The rate constant for hydride transfer from NADPH to flavin was lowest for R174A (k (red) = 8.82 s(-1) versus 22.63 s(-1) for the wild type), which also exhibi ted tertiary structure perturbation, as evidenced by alterations in CD and fluorescence spectra. Molecular modelling indicated that movement of the C- terminal tryptophan (W248) of FLDR is necessary to permit close approach of the nicotinamide ring of NADPH to the flavin. The positions of NADPH phosp hates in the modelled structure are consistent with the kinetic data, with R174 and R184 located close to the adenosine ribose 2'-phosphate group, and R144 likely to interact with the nicotinamide ribose 5'-phosphate group.