Rapid-mix and chemical quench studies of ferredoxin-reduced stearoyl-acyl carrier protein desaturase.

Stearoyl-ACP Delta9 desaturase (Delta9D) catalyzes the NADPH- and O(2)-dependent insertion of a cis double bond between the C9 and C10 positions of stearoyl-ACP (18:0-ACP) to produce oleoyl-ACP (18:1-ACP). This work revealed the ability of reduced [2Fe-2S] ferredoxin (Fd) to act as a catalytically competent electron donor during the rapid conversion of 18:0-ACP into 18:1-ACP. Experiments on the order of addition for substrate and reduced Fd showed high conversion of 18:0-ACP to 18:1-ACP (approximately 95% per Delta9D active site in a single turnover) when 18:0-ACP was added prior to reduced Fd. Reactions of the prereduced enzyme-substrate complex with O(2) and the oxidized enzyme-substrate complex with reduced Fd were studied by rapid-mix and chemical quench methods. For reaction of the prereduced enzyme-substrate complex, an exponential burst phase (k(burst) = 95 s(-1)) of product formation accounted for approximately 90% of the turnover expected for one subunit in the dimeric protein. This rapid phase was followed by a slower phase (k(linear) = 4.0 s(-1)) of product formation corresponding to the turnover expected from the second subunit. For reaction of the oxidized enzyme-substrate complex with excess reduced Fd, a slower, linear rate (k(obsd) = 3.4 s(-1)) of product formation was observed over approximately 1.5 turnovers per Delta9D active site potentially corresponding to a third phase of reaction. An analysis of the deuterium isotope effect on the two rapid-mix reaction sequences revealed only a modest effect on k(burst) ((D)k(burst) approximately 1.5) and k(linear) (D)k(linear) approximately 1.4), indicating C-H bond cleavage does not contribute significantly to the rate-limiting steps of pre-steady-state catalysis. These results were used to assemble and evaluate a minimal kinetic model for Delta9D catalysis.