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

Abstract

Tyr25 is a ligand to the active site d 1 heme in as isolated, oxidized cytochrome cd 1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr 25 from the d 1 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 d 1 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 Tyr 25 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 cd 1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d 1 heme ring for anions in the absence of the steric barrier presented by Tyr 25 .

Probing the Unusual Oxidation/Reduction Behavior ofParacoccus pantotrophusCytochromecd1Nitrite Reductase by Replacing a Switchable Methionine Heme Iron Ligand with Histidine

“…Previous experiments performed on Y25S variant P. pantotrophus cytochrome cd 1 , which has His/Met ligands to both the oxidized and reduced c heme in solution, demonstrated that reduced pseudoazurin was able to reduce the protein (31). In contrast, we found that pseudoazurin failed to reduce M106H cytochrome cd 1 .…”

“…It is well documented that the average midpoint redox potential of a His/Met coordinated c heme is commonly ∼200 mV in excess of a bishistidinyl coordinated c heme because methionine is more effective than histidine at stabilizing the reduced c heme iron (19,40). This is highlighted by the observation that, in this work, reduced pseudoazurin could not reduce the bishistidinyl M106H variant protein; however, it was able to reduce a Y25S variant with His/Met c heme coordination due to the higher midpoint potential of a His/Met c heme center (31).…”

Pseudoazurin Dramatically Enhances the Reaction Profile of Nitrite Reduction by Paracoccus pantotrophus Cytochrome cd1 and Facilitates Release of Product Nitric Oxide

“…2). Notably, no d 1 heme signals other than the trace of Fe(II)-NO were observed in the EPR spectrum of cd 1 -X reacted with oxidized pseudoazurin, fully consistent with the final product being the (EPR-silent (25,27)) all-ferric state of the enzyme with nitrite bound.…”

“…To obtain the spectra of these complexes, fully reduced cytochrome cd 1 was mixed with nitrite in a stopped-flow apparatus, and spectra were recorded over a period of 2 s with a minimum data interval of 2 ms. cd 1 -X was either allowed to decay in the intense light of the diode array spectrophotometer (v) or was mixed with initially oxidized pseudoazurin (vi). Rows vii-ix refer to standard (model) cytochrome cd 1 complexes used in the assignment of spectra described in this work; vii and viii describe spectra of the Y25S variant of P. pantotrophus cytochrome cd 1 (27,34 cytochrome cd 1 ( Table 1). The final product of the reaction between pseudoazurin and cd 1 -X at pH 7.0 had a visible absorption peak at 641 nm, and when the same experiment was repeated at pH 6.0, the peak was at 639 nm.…”

“…The final product of the reaction between pseudoazurin and cd 1 -X at pH 7.0 had a visible absorption peak at 641 nm, and when the same experiment was repeated at pH 6.0, the peak was at 639 nm. Model spectra for both nitrite and NO bound to fully oxidized cytochrome cd 1 were obtained using the Y25S variant of P. pantotrophus cytochrome cd 1 , which, unlike the "as isolated" oxidized wild type enzyme, is fully competent for exogenous ligand binding (25)(26)(27). The d 1 heme peak in the spectrum of nitrite bound to all-ferric Y25S enzyme varied from 639 to 642 nm as the pH shifted from 6.0 to 7.0 (Table 1).…”