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J. Biol. Chem., Vol. 280, Issue 28, 26073-26079, July 15, 2005

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Y25S Variant of Paracoccus pantotrophus Cytochrome cd₁ Provides Insight into Anion Binding by d₁ Heme and a Rare Example of a Critical Difference between Solution and Crystal Structures^*

Richard S. Zajicek, Myles R. Cheesman, Euan H. J. Gordon¶||, and Stuart J. Ferguson**

From the Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom, School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom, and ^¶Department of Chemistry and Bioscience, Chalmers University of Technology, SE-412 96 Gothenburg, Sweden

Tyr²⁵ is a ligand to the active site d₁ heme in as isolated, oxidized cytochrome cd₁ nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr²⁵ from the d₁ heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d₁ heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr²⁵ is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd₁ is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d₁ heme ring for anions in the absence of the steric barrier presented by Tyr²⁵.

Received for publication, February 18, 2005 , and in revised form, May 13, 2005.

^* This work was supported in part by Grant C19430 and a studentship (to R. S. Z.) from the Biotechnology and Biological Sciences Research Council. 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.

^|| Supported in part by European Union Biotechnology Structural Biology Project BIO4 CT96-0281.

^** To whom correspondence should be addressed: Dept. of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom. Tel.: 01865275240; Fax: 01865275259; E-mail: stuart.ferguson{at}bioch.ox.ac.uk.

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