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,Stuart J. Ferguson,Myles R. Cheesman,Euan H.J. Gordon

doi:10.1074/jbc.m501890200

Abstract

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.

Highlights

There are two strikingly different forms of bacterial nitrite reductase used in denitrification to catalyze the one-electron reduction of nitrite (NO2Ϫ) to nitric oxide (NO) and water, namely, cytochrome cd[1] (NirS) and the copper-containing enzyme NirK
The Soret band of the Y25S cytochrome cd[1] spectrum is red shifted from 406 nm for the wild type to 410 nm, a value consistent with, but not reliably diagnostic for, His/Met coordination of the c heme as in other cytochromes cd[1] (2, 13, 14), including activated P. pantotrophus cytochrome cd[1] (6, 15)
MCD and EPR spectroscopies categorically identify the axial ligands at the c heme of P. pantotrophus Y25S cytochrome cd[1] as His/Met in solution at room temperature and the majority population at two different pH values at very low temperature

Summary

Introduction

There are two strikingly different forms of bacterial nitrite reductase used in denitrification to catalyze the one-electron reduction of nitrite (NO2Ϫ) to nitric oxide (NO) and water, namely, cytochrome cd[1] (NirS) and the copper-containing enzyme NirK. Subsequent to the initial spectroscopic and crystallographic characterization of the enzyme, it was discovered that significant catalytic activity of P. pantotrophus cytochrome cd[1] required activation by reduction (4) This could be understood in terms of the bis-histidinyl ligands to the oxidized c heme resulting in this center having an E°Ј of approximately ϩ60 mV. The protein still had bishistidinyl coordination at the c heme, and the serine had effectively replaced Tyr[25] in the d1 heme pocket, but a sulfate ion (presumably from the precipitant) was bound to the d1 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 d1 heme permitting nitrite to bind and subsequently raise the reduction potential of that heme (9). Additional spectroscopic studies of the Y25S protein have, as described in the present work, unexpectedly undermined this conclusion

Methods

Results

Conclusion