Optical, EPR and Mossbauer spectroscopic studies on the NO derivatives of cytochrome cd1 from Thiobacillus denitrificans

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Abstract
We have used optical, EPR and Mossbauer spectroscopies to study the formation of heme-NO complex upon the addition of nitrite to reduced cytochrome cd1 from Thiobacillus denitrificans. The reduced d1 heme binds NO under both alkaline and acidic conditions, but the binding of NO to the reduced c heme was strongly pH-dependent. The Mossbauer data showed umambiguously that at pH 7.6 the c heme does not complex NO, whereas at pH 5.8 approximately half of the reduced c heme binds NO. This observation was confirmed by EPR studies, which showed that the spin concentration of the heme-NO EPR signal increased from 2 spins/molecule at pH 8.0 to approximately 3 spins/molecule at pH 5.8. Optical absorption study also showed strong pH dependence in the binding of NO to the reduced c heme. We have also analyzed the Mossbauer spectra of the ferrous d1 heme-NO complex using a spin-Hamiltonian formalism. The magnetic hyperfine coupling tensor was found to be consistent with the unpaired electron residing on a .sigma. orbital.