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Approaches to a reaction mechanism for soybean ferric leghemoglobin reductase
Abstract
A protein with ferric leghemoglobin reductase (FLbR) activity was isolated and purified from soybean nodules. The gene encoding FLbR was cloned, sequenced and the recombinant FLbR was expressed in Escherichia coli. In this dissertation the sequence and size of the cloned insert DNA were determined to confirm that the recombinant DNA is FLbR gene. Western blot analysis of the protein and the kinetic properties ($K\sb{\rm m}$ and $k\sb{\rm cat}$) of the enzyme support that the recombinant protein is soybean FLbR. The enzymatic activities and kinetic properties were compared with dihydrolipoamide dehydrogenases (DHLipDHs). FLbR has multiple activities but it has the highest affinity and specificity for the ferric state of leghemoglobin (Lb). The evidence supports the postulation that FLbR functions to reduce the ferric state of Lb. FLbR has a disulfide group in the active site of the enzyme and a two-electron transfer dominates in the reduction of the ferric state of Lb as shown by the inhibition of FLbR activity by disulfide-binding compounds. The mid-point potential (E$\sb{\rm m}$) of FLbR were determined by spectrotitration to be $-0.294$ V for disulfide/dithiol and $-0.318$ V for the FAD/FADH$\sb2$. From the spectral studies and the E$\sb{\rm m}$ values, I postulated that two electrons are transferred from NADH to FAD, to the disulfide group, and then to a substrate, Lb$\sp{3+}$. The possible mechanisms of reaction were proposed using a known mechanism of DHLipDHs as a model reaction.
Subject Area
Biochemistry|Molecular biology|Botany
Recommended Citation
Kim, Hyun-Mi, "Approaches to a reaction mechanism for soybean ferric leghemoglobin reductase" (1996). ETD collection for University of Nebraska-Lincoln. AAI9700093.
https://digitalcommons.unl.edu/dissertations/AAI9700093