Structure and Kinetic Properties of Paracoccus pantotrophus Cytochrome cd1 Nitrite Reductase with the d1 Heme Active Site Ligand Tyrosine 25 Replaced by Serine

Abstract

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.

Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases

“…Actually, in bacterial enzymes containing more than one domain, such as bacterial cytochrome c peroxidase and nitrite reductase cytochrome cd 1 , it has been observed that reduction of the redox center in one of the domains implies a structural change in the protein [62][63][64][65]. Therefore, similar behavior cannot be ruled out for this enzyme and further experiments are currently in progress in our laboratory to test this hypothesis.…”

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

“…This was to be expected, following the loss of Tyr 25 , which puts the d 1 heme in a state of high/low spin thermal equilibrium (2). Upon removal of Tyr 25 , there was only a signal associated with a low spin d 1 heme species (2,9). A 1.4 Å structure of this variant protein showed only very localized changes in the immediate vicinity of residue 25 (9).…”

“…The protein still had bishistidinyl coordination at the c heme, and the serine had effectively replaced Tyr 25 in the d 1 heme pocket, but a sulfate ion (presumably from the precipitant) was bound to the d 1 heme rather than the ϪOH of Ser 25 . Kinetic assays revealed this enzyme did not require pre-reduction for maximal substrate turnover, unlike wild type cytochrome cd 1 (4,9), possibly as a result of the increased accessibility of the d 1 heme permitting nitrite to bind and subsequently raise the reduction potential of that heme (9). The latter might allow electron transfer from a donor protein even with an energetically uphill initial step due to His/His coordination of the c heme (9,10).…”

“…Upon removal of Tyr 25 , there was only a signal associated with a low spin d 1 heme species (2,9). A 1.4 Å structure of this variant protein showed only very localized changes in the immediate vicinity of residue 25 (9). The protein still had bishistidinyl coordination at the c heme, and the serine had effectively replaced Tyr 25 in the d 1 heme pocket, but a sulfate ion (presumably from the precipitant) was bound to the d 1 heme rather than the ϪOH of Ser 25 .…”

“…Tyr 25 was subsequently mutated to a serine residue (9) to probe the consequences of the loss of this tyrosine ligand on * This work was supported in part by Grant C19430 and a studentship (to R. S. Z.)…”