ABSTRACT
Paracoccus pantotrophus cytochrome cd(1) is a physiological nitrite reductase and an in vitro hydroxylamine reductase. The oxidised "as isolated" form of the enzyme has bis-histidinyl coordinated c-heme and upon reduction its coordination changes to histidine/methionine. Following treatment of reduced enzyme with hydroxylamine, a novel, oxidised, conformer of the enzyme is obtained. We have devised protocols for freeze-quench near-ir-MCD spectroscopy that have allowed us to establish unequivocally the c-heme coordination of this species as His/Met. Thus it is shown that the catalytically competent, hydroxylamine reoxidised, form of P. pantotrophus cytochrome cd(1) has different axial ligands to the c-heme than "as isolated" enzyme.
Subject(s)
Cytochromes/chemistry , Cytochromes/metabolism , Nitrite Reductases/chemistry , Nitrite Reductases/metabolism , Paracoccus/enzymology , Cytochrome c Group , Electron Spin Resonance Spectroscopy/methods , Freezing , Oxidation-Reduction , Protein Conformation , Spectrophotometry, Infrared/methodsABSTRACT
The alpha- and beta-anomers of D-cellobiose were resolved by 1H NMR spectroscopy. Addition of cellobiose dehydrogenase purified from the white-rot P. chrysosporium led to selective conversion of beta-D-cellobiose. The product was identical to cellobionolactone as synthesized from Ca-cellobionate. Overnight incubation of the product led to an altered NMR spectrum, which was also obtained by incubation of cellobionolactone. The new spectrum matched that for Ca-cellobionate. The instability of cellobionolactone explains the detection of cellobionic acid as product in earlier studies.
