Figure 3.
Comparison of the Redox and Molecular Properties of the Wild-Type and p.Glu493Val and p.Arg201del Mutants of AIF
(A) Anaerobic titration of AIFE493V with NADH. Similar to the wild-type protein, the p.Glu493Val variant binds NADH tightly and produces a FADH−-NAD charge-transfer complex (CTC) absorbing in the long-wavelength region. Inset shows that an equimolar amount of NADH is required to fully reduce FAD.
(B) Kinetics of AIF reduction with NADH. Proteins were mixed with NADH in a stopped flow spectrophotometer, and reduction of FAD was monitored at 452 nm. The derived kinetic parameters are given in Table 1.
(C) Kinetics of oxidation of NADH-reduced AIF. Proteins were reduced with a 4-fold excess of NADH before exposure to atmospheric oxygen. Flavin oxidation was monitored at 452 nm.
(D) Effect of pH on the kinetics of AIF reduction with NADH.
(E) Comparison of DNA binding by wild-type and mutant AIF. Equal amounts of protein (100 μg) were incubated for 15 min with 250 ng of 100 bp DNA ladder (New England Biolabs) in the absence and presence of a 20-fold excess of NADH. After separation on a 2% agarose gel, DNA was visualized with ethidium bromide. Lane 1 is a control (DNA only).