Effects of hydrogen peroxide upon nicotinamide nucleotide metabolism in Escherichia coli: changes in enzyme levels and nicotinamide nucleotide pools and studies of the oxidation of NAD(P)H by Fe(III).
DNA is damaged in vivo by the Fenton reaction mediated by Fe2+ and cellular reductants such as NADH, which reduce Fe3+ to Fe2+ and allow the recycling of iron. To study the response of Escherichia coli to such cycling, the activities of several enzymes involved in nicotinamide nucleotide metabolism were measured following an H2O2 challenge. NADPH-dependent peroxidase, NADH/NADP+ transhydrogenase, and glucose-6-phosphate dehydrogenase were most strongly induced, increasing 2.5-3-fold. In addition, the cellular ratios of NADPH to NADH increased 6- or 92-fold 15 min after exposure to 0.5 or 5 mm H2O2, respectively. In vitro, NADH was oxidized by Fe3+ up to 16-fold faster than NADPH, despite their identical reduction potentials. To understand this rate difference, the interactions of Fe3+ and Ga3+ with NAD(P)H were examined by 1H, 13C, and 31P NMR spectroscopy. Association with NADH occurred primarily with adenine at N7 and the amino group, but for NADPH, strong metal interactions also occurred at the 2'-phosphate group. Interaction of M3+ (Fe3+ or Ga3+) with the adenine ring would bring it into close proximity to the redox-active nicotinamide ring in the folded form of NAD(P)H, but interaction of M3+ with the 2'-phosphate group would avoid this close contact. In addition, as determined by absorbance spectroscopy, the energy of the charge-transfer species was significantly higher for the Fe3+.NADPH complex than for the Fe3+.NADH complex. We therefore suggest that upon exposure to H2O2 the NADH pool is depleted, and NADPH, which is less reactive with Fe3+, functions as the major nicotinamide nucleotide reductant.
Brumaghim
Julia L
JL
Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720-3202, USA.
Li
Ying
Y
Henle
Ernst
E
Linn
Stuart
S
D004798:Enzymes / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D005708:Gallium / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism / Q000502:physiology; D009537:Nicotinamide Mononucleotide / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D013057:Spectrum Analysis; D017186:Titrimetry
D004798:Enzymes; D009537:Nicotinamide Mononucleotide; D009249:NADP; D006861:Hydrogen Peroxide; D005708:Gallium; D007501:Iron