UV–Vis and EPR analyses of CobDH. (a) Anaerobic titration of oxidized CobDH with titanium(III) citrate as a reductant. The absorbance maximum of cob(III)alamin at 540 nm decreases with increasing amounts of added reductant; the absorbance of cob(II)alamin (474 nm) and cob(I)alamin (388 nm) increase correspondingly. The inset shows a plot of the percentage of CobDH in each of the three possible oxidation states of its cofactor during titration with titanium(III) citrate. The respective oxidation-state percentages were calculated as described in §2. The cob(III)alamin percentage in CobDH decreases from approximately 53 to 22%, whereas the percentages of cob(II)alamin and cob(I)alamin increase from 37 to 60% and from 8 to 18%, respectively. (b) The oxidized EPR sample refers to CobDH as purified. The cob(III)alamin–superoxide complex signal has a g value of 2.0 (magnetic field of 3400 G; upper spectra). After sample reduction with 5 mM sodium dithionite the spectrum shows the presence of the base-off/His-on form with a main feature at g = 2.26 (magnetic field of 2900 G; lower spectra). Also present is hyperfine splitting of the signal, which arises from the interaction between the cob(II)alamin and its β-axial coordinated N atom from the His102 side chain.