FIGURE 2.

Purified B. megaterium CbiH₆₀ UV-visible and EPR spectra. A, SDS gel showing purified CbiH₆₀ proteins used in this study. The molecular mass markers are shown under lane M; lane WT contains ∼3 μg of CbiH₆₀; lane C402A contains ∼1.5 μg of C402A CbiH₆₀; and lane C443A contains ∼1.5 μg of C402A CbiH₆₀. B, UV-visible spectra of 50 μm CbiH₆₀ as purified (solid line) and after reduction with excess dithionite (dotted line). C, UV-visible spectra of 50 μm CbiH₆₀ exposed to air and scanned every 10 min. Arrows show a loss of absorbance between 300 and 450 nm, indicating the loss of the [4Fe-4S] cluster. D, EPR of 200 μm (12 mg ml⁻¹) CbiH₆₀ (buffer-exchanged in Buffer H), reduced with excess dithionite (10 mm) and frozen in liquid nitrogen. From the intensity of the signal, ∼20–25 μm (∼10%) of reduced [4Fe-4S]¹⁺is present. The [4Fe-4S]¹⁺ spectrum of CbiH₆₀ was recorded at 12 K, employing a microwave frequency of 9.383984 GHz, microwave power of 1 milliwatt, a modulation frequency of 100 kHz, and a modulation amplitude of 5 G. A spectrum of CbiH₆₀ was also recorded at 70 K, which resulted in a loss of the [4Fe-4S]¹⁺ signal. This indicated t1hat the iron-sulfur center was of the [4Fe-4S] class.