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. 2006 May 31;128(21):6766-7.
doi: 10.1021/ja058822p.

Catalytic reduction of NO to N2O by a designed heme copper center in myoglobin: implications for the role of metal ions

Xuan Zhao  1 , Natasha Yeung, Brandy S Russell, Dewain K Garner, Yi Lu
Affiliations

Catalytic reduction of NO to N2O by a designed heme copper center in myoglobin: implications for the role of metal ions

Xuan Zhao et al. J Am Chem Soc. .

Abstract

The effects of metal ions on the reduction of nitric oxide (NO) with a designed heme copper center in myoglobin (F43H/L29H sperm whale Mb, CuBMb) were investigated under reducing anaerobic conditions using UV-vis and EPR spectroscopic techniques as well as GC/MS. In the presence of Cu(I), catalytic reduction of NO to N2O by CuBMb was observed with turnover number of 2 mol NO.mol CuBMb-1.min-1, close to 3 mol NO.mol enzyme-1.min-1 reported for the ba3 oxidases from T. thermophilus. Formation of a His-heme-NO species was detected by UV-vis and EPR spectroscopy. In comparison to the EPR spectra of ferrous-CuBMb-NO in the absence of metal ions, the EPR spectra of ferrous-CuBMb-NO in the presence of Cu(I) showed less-resolved hyperfine splitting from the proximal histidine, probably due to weakening of the proximal His-heme bond. In the presence of Zn(II), formation of a five-coordinate ferrous-CuBMb-NO species, resulting from cleavage of the proximal heme Fe-His bond, was shown by UV-vis and EPR spectroscopic studies. The reduction of NO to N2O was not observed in the presence of Zn(II). Control experiments using wild-type myoglobin indicated no reduction of NO in the presence of either Cu(I) or Zn(II). These results suggest that both the identity and the oxidation state of the metal ion in the CuB center are important for NO reduction. A redox-active metal ion is required to deliver electrons, and a higher oxidation state is preferred to weaken the heme iron-proximal histidine toward a five-coordinate key intermediate in NO reduction.

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Figures

Figure 1
Figure 1
(A) The active site of a computer model of CuBMb; (B) UV-Vis spectra of ferrous CuBMb-NO in the absence of metal ions (dotted line); in the presence of Cu(I) (dashed line) and in the presence of Zn(II) (solid line); EPR spectra of (C) ferrous-CuBMb-NO and (D) ferrous-CuBMb-15NO in the absence of metal ions; (E) ferrous-CuBMb-NO and (F) ferrous-CuBMb- 15NO in the presence of copper; (G) ferrous-CuBMb-NO and (H) ferrous-CuBMb- 15NO in the presence of Zn(II). Samples were recorded in 20 mM Tris pH 8 at 45 K and 0.2 mW power; microwave frequency, 9.050 GHz.
Figure 2
Figure 2
GC/MS chromatogram of NO reduction by CuBMb and Cu2+The GC peaks have been normalized.
See this image and copyright information in PMC

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