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J. Biol. Chem., Vol. 278, Issue 43, 42495-42504, October 24, 2003

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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)^*

Julia L. Brumaghim, Ying Li, Ernst Henle¶, and Stuart Linn||

From the Division of Biochemistry and Molecular Biology, University of California, Berkeley, California 94720-3202

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.

Received for publication, June 13, 2003 , and in revised form, August 7, 2003.

^* This work was supported by in part by National Institutes of Health Grants RO1GM19020 and P30ES01986. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Tables S1 and S2 and Figs. S1, S2, and S3.

Supported by National Institutes of Health Training Grant T32ES07075. Present address: Dept. of Chemistry, Clemson University, 481 Hunter Laboratories, Clemson University, Clemson, SC 29634.

Present address: Pfizer, Inc., 4125 Sorrento Valley Blvd., San Diego, CA 92121.

^¶ Present address: Scimagix, Inc., 2855 Campus Dr., Suite 100, San Mateo, CA 94403.

^|| To whom correspondence should be addressed: Division of Biochemistry and Molecular Biology, Barker Hall, University of California, Berkeley, CA 94720-3202. Tel.: 510-642-7583; Fax: 510-643-3388; E-mail: slinn{at}socrates.berkeley.edu.

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