The 1.4-Å crystal structure of the oxidized state of a Y25S variant of cytochrome cd 1 nitrite reductase from Paracoccus pantotrophus is described. It shows that loss of Tyr 25 , a ligand via its hydroxy group to the iron of the d 1 heme in the oxidized (as prepared) wild-type enzyme, does not result in a switch at the c heme of the unusual bishistidinyl coordination to the histidine/methionine coordination seen in other conformations of the enzyme. The Ser 25 side chain is seen in two positions in the d 1 heme pocket with relative occupancies of ϳ7:3, but in neither case is the hydroxy group bound to the iron atom; instead, a sulfate ion from the crystallization solution is bound between the Ser 25 side chain and the heme iron. Unlike the wild-type enzyme, the Y25S mutant is active as a reductase toward nitrite, oxygen, and hydroxylamine without a reductive activation step. It is concluded that Tyr 25 is not essential for catalysis of reduction of any substrate, but that the requirement for activation by reduction of the wild-type enzyme is related to a requirement to drive the dissociation of this residue from the active site. The Y25S protein retains the d 1 heme less well than the wild-type protein, suggesting that the tyrosine residue has a role in stabilizing the binding of this cofactor.

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