Resonance Raman studies of the stoichiometric catalytic turnover of a substrate-stearoyl-acyl carrier protein delta(9) desaturase complex.
Resonance Raman spectroscopy has been used to study the effects of substrate binding (stearoyl-acyl carrier protein, 18:0-ACP) on the diferric centers of Ricinus communis 18:0-ACP Delta(9) desaturase. These studies show that complex formation produces changes in the frequencies of nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) consistent with a decrease in the Fe-O-Fe angle from approximately 123 degrees in the oxo-bridged diferric centers of the as-isolated enzyme to approximately 120 degrees in oxo-bridged diferric centers of the complex. Analysis of the shifts in nu(s)(Fe-O-Fe) and nu(as)(Fe-O-Fe) as a function of 18:0-ACP concentration also suggests that 4e(-)-reduced Delta9D containing two diferrous centers has a higher affinity for 18:0-ACP than resting Delta9D containing two diferric centers. Catalytic turnover of a stoichiometric complex of 18:0-ACP and Delta9D was used to investigate whether an O-atom from O(2) would be incorporated into a bridging position of the resultant mu-oxo-bridged diferric centers during the desaturation reaction. Upon formation of approximately 70% yield of 18:1-ACP product in the presence of (18)O(2), no incorporation of an (18)O atom into the mu-oxo bridge position was detected. The result with 18:0-ACP Delta(9) desaturase differs from that obtained during the tyrosyl radical formation reaction of the diiron enzyme ribonucleotide reductase R2 component, which proceeds with incorporation of an O-atom from O(2) into the mu-oxo bridge of the resting diferric site. The possible implications of these results for the O-O bond cleavage reaction and the nature of intermediates formed during Delta9D catalysis are discussed.
Lyle
K S
KS
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison, Wisconsin 53706-1544, USA.
Möenne-Loccoz
P
P
Ai
J
J
Sanders-Loehr
J
J
Loehr
T M
TM
Fox
B G
BG
D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D010947:Plants, Toxic; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012278:Ricinus / Q000201:enzymology; D013059:Spectrum Analysis, Raman; D013379:Substrate Specificity
D000213:Acyl Carrier Protein; D011994:Recombinant Proteins; D006899:Mixed Function Oxygenases; C018747:acyl-(acyl-carrier-protein)desaturase