Mike M. Chen et al.
COMMUNICATIONS
substrate for 2 min before the addition of 0.27 mL of pre-sa-
turated 0.1M phosphate buffer, pH 8.0. The head-space was
then pressurized to 20 psi with the gaseous alkane. All reac-
tions were initated by the addition of the oxidant and al-
lowed to proceed for 10 min at 258C. Reactions with liquid
alkanes were extracted with 150 mL of chloroform and ana-
lyzed by GC-FID with the addition of 2-nonanol as an inter-
nal standard. Reactions with gaseous alkanes were quenched
with 20 mL of 3.0M HCl and neutralized with 75 mL of 1.0M
phosphate buffer, pH 8.0 to precipitate the enzymes in solu-
tion. After centrifugation at 14,000 g for 2 min, the resulting
solution was analyzed by GC-MS.
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A6 in vitro Hydroxylation Reactions
For the in vitro A6 alkane hydroxylation reactions, purified
reductase components, ferredoxin reductase (fdrA6) and fer-
redoxin (fdxA6) were quantified using known extinction co-
efficients for their FAD and [Fe2-S2] cofactors.[21] A ratio of
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Acknowledgements
This work was supported by the Chemical Sciences, Geo-
sciences and Biosciences Division, Office of Basic Science,
U.S.Department of Energy grant DE-FG02-06ER15762. We
thank Dr. Nathan Dalleska for assistance with gas chroma-
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Adv. Synth. Catal. 2012, 354, 964 – 968