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J. Biol. Chem., Vol. 281, Issue 24, 16340-16346, June 16, 2006

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A Random-sequential Mechanism for Nitrite Binding and Active Site Reduction in Copper-containing Nitrite Reductase^*

Hein J. Wijma¹, Lars J. C. Jeuken, Martin P. Verbeet, Fraser A. Armstrong^¶, and Gerard W. Canters²

From the Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands, the Institute of Molecular Biophysics, University of Leeds, Leeds LS2 9JT, United Kingdom, and the ^¶Inorganic Chemistry Laboratory, Oxford University, South Parks Road, Oxford OX1 3QR, United Kingdom

The homotrimeric copper-containing nitrite reductase (NiR) contains one type-1 and one type-2 copper center per monomer. Electrons enter through the type-1 site and are shuttled to the type-2 site where nitrite is reduced to nitric oxide. To investigate the catalytic mechanism of NiR the effects of pH and nitrite on the turnover rate in the presence of three different electron donors at saturating concentrations were measured. The activity of NiR was also measured electrochemically by exploiting direct electron transfer to the enzyme immobilized on a graphite rotating disk electrode. In all cases, the steady-state kinetics fitted excellently to a random-sequential mechanism in which electron transfer from the type-1 to the type-2 site is rate-limiting. At low [NO^–₂] reduction of the type-2 site precedes nitrite binding, at high [NO^–₂] the reverse occurs. Below pH 6.5, the catalytic activity diminished at higher nitrite concentrations, in agreement with electron transfer being slower to the nitrite-bound type-2 site than to the water-bound type-2 site. Above pH 6.5, substrate activation is observed, in agreement with electron transfer to the nitrite-bound type-2 site being faster than electron transfer to the hydroxyl-bound type-2 site. To study the effect of slower electron transfer between the type-1 and type-2 site, NiR M150T was used. It has a type-1 site with a 125-mV higher midpoint potential and a 0.3-eV higher reorganization energy leading to an 50-fold slower intramolecular electron transfer to the type-2 site. The results confirm that NiR employs a random-sequential mechanism.

Received for publication, February 21, 2006 , and in revised form, April 12, 2006.

^* 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 supplemental Table S1, Fig. S1, equations, and a supplemental scheme.

¹ Present address: Duke University, Medical Center, Dept. of Biochemistry, Durham, NC 27710.

² To whom correspondence should be addressed. E-mail: Canters{at}chem.leidenuniv.nl.

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