Mechanism of Reduction of Quinones by Trypanosoma congolense Trypanothione Reductase

Narimantas K. Cenas, David Arscott, Charles H. Williams, John S. Blanchard

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Abstract

A number of quinones were analyzed as substrates for trypanothione reductase from Trypanosoma congolense, an enzyme responsible for the protection of trypanosomes against oxidative stress. Using NADPH as substrate, the maximal rate of the steady-state reaction at pH 7.5 was between 24 and 1.6 s−1 for 11 quinone substrates. The biomolecular steady-state rate constants for quinone reduction, V/Km, ranged from 240 to 1.9 × 105 M−1 s−1 and their logarithms exhibited a hyperbolic dependence on the one-electron-reduction potentials of the quinone substrate. The addition of NADP+ stimulated these rates, with V/Km values increasing with an increasing NADP+/NADPH ratio. The results of alkylation of the cysteine residue in the two-electron-reduced enzyme by iodoacetamide indicate that these residues are not primarily involved in the reduction of these quinones. Single-electron reduction of benzoquinone constitutes 40% of the total electron transfer from NADPH to quinone in the absence of NADP+, and increases to 80% at NADP+/NADPH ratios greater than 10. These steady-state results were confirmed in pre-steadystate rapid reaction experiments. The rate of reduced enzyme oxidation by 1,4-benzoquinone is approximately 100 times faster in the presence of NADP+ than in its absence. The reactivities of various pyridine nucleotide liganded forms of EH2 for quinone reduction are presumably affected by the electron density at FAD. We suggest that one-electron reduction of quinones occurs at a site distinct from the two active sites involved in hydride ion transfer and disulfide reduction.

Original languageEnglish (US)
Pages (from-to)2509-2515
Number of pages7
JournalBiochemistry
Volume33
Issue number9
DOIs
StatePublished - Mar 1 1994

ASJC Scopus subject areas

  • Biochemistry

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