Table 2.

Biochemical characterization of CntA wild-type and variant proteins^a

Mutation	Function	Relative l-carnitine depletion activity (%)	Relative TMA formation (%)	Iron content (nmol protein−1)	Sulfur content (nmol protein−1)	EPR signal for [Fe]	EPR signal for [2Fe-2S]
C86A	[2Fe-2S] coordinating	<5	–	0.12 ± 0.03	0.35 ± 0.11	ND	ND
H88A	[2Fe-2S] coordinating	<5	ND	0.06 ± 0.03	0.64 ± 0.21	Weak	ND
C106A	[2Fe-2S] coordinating	<5	–	0.03 ± 0.02	0.03 ± 0.02	Weak	ND
H109A	[2Fe-2S] coordinating	<5	–	0.03 ± 0.02	0.67 ± 0.08	ND	ND
E205Q	Intersubunit electron transfer	<5	–	2.78 ± 0.10	1.62 ± 0.14	Weak	WT
E205D	Intersubunit electron transfer	<5	ND	2.53 ± 0.15	1.53 ± 0.07	Weak	WT
H208A	[Fe] coordinating	<5	–	2.35 ± 0.11	2.08 ± 0.24	ND	WT
H213A	[Fe] coordinating	<5	ND	2.00 ± 0.11	1.39 ± 0.15	ND	WT
D323A	[Fe] coordinating	<5	–	2.71 ± 0.15	1.76 ± 0.28	ND	WT

^aTable summarizes biochemical and spectroscopic properties of CntA variants. The specific activity of the l-carnitine depletion assay (771 ± 68 nmol min⁻¹ mg⁻¹) and of the enzymatic TMA formation (determined by gas chromatography) for the wild-type protein was set as 100% (compared to experimental procedures). ND, TMA formation not detectable. EPR signals for the [Fe] center (samples as purified) and the [2Fe-2S] cluster (samples after dithionite reduction) were classified. EPR signals comparable with the wild type are indicated as WT, substantially reduced EPR signals (<10% of wild-type signal) are indicated as weak, and the absence of a detectable EPR signal was indicated as not detectable (ND). <5 indicates enzymatic activity below the detection limit of the employed assay. –, not performed.