Table 1.

Steady state activity of CytcO-SMA native nanodiscs and of CytcO in the S. cerevisiae mitochondrial membrane^*.

Sample	Ratio heme aa3/protein, nmole/mg#)	CytcO activity, e−/s
		No DDM	+0.05% DDM
CytcO-SMA	0.9 ± 0.4 (n = 4)	130 ± 20 (n = 3)	300 ± 40 (n = 3)
	1.1 ± 0.3 (n = 5)	90 ± 20 (n = 3)	190 ± 40 (n = 3)
Mitochondria	0.08 ± 0.04 (n = 6)	240 ± 20 (n = 6)	700 ± 300 (n = 4)

^*For CytcO in SMA native nanodiscs, the heme content was calculated either from the difference spectra “reduced-minus-as purified” or from absolute reduced spectrum (shown in italic, using ε = 39 mM⁻¹ cm⁻¹ [31] at 604 nm). Accordingly, two different values are given for the steady-state activity, which is normalized to the CytcO concentration. Because the isolated CytcO was slightly reduced in the “as purified” nanodiscs, the values obtained from the difference spectra yielded an underestimation of the heme concentration.

^#The ratio of heme aa₃ concentration over the protein amount (see “Materials and Methods” section) is a measure of the CytcO purity. For 100% pure CytcO, with a molecular weight of 200 kDa, the ratio is ${\frac{1}{2\cdot {10}^5}}^{\text{mole}/\text{g}}=5^{\text{nmole}/\text{mg}}$. For detergent-purified CytcO we typically obtained a ratio of ~1. The data show that isolation of the CytcO native nanodiscs resulted in an enrichment of the CytcO by a factor of ~10.