Effect of Substrate on the Diiron(III) Site in Stearoyl Acyl Carrier Protein Δ⁹-Desaturase as Disclosed by Cryoreduction Electron Paramagnetic Resonance/Electron Nuclear Double Resonance Spectroscopy

Abstract

The diiron center in stearoyl-acyl carrier protein (ACP) desaturase (DS) from castor plant Ricinus communis catalyzes the dioxygen- and NADPH-dependent introduction of a cis double bond between C9 and C10 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77 K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center (or [DS(ox)](mv)) that is trapped in the conformation of the diferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cryoreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states: the mu-oxo (major)- and mu-hydroxo (minor)-bridged diiron centers. ENDOR studies show that in the dominant oxo-bridged diferric state each iron(III) coordinates a histidine and a water along with other ligands. Samples containing stoichiometric amounts of stearoyl-ACP show pronounced changes in the EPR and (1)H ENDOR spectra of cryoreduced DS. EPR spectra of the cryoreduced DS-substrate complex reveal two distinct substates of the parent. EPR and ENDOR studies show that both major conformers of the diferric cluster have a mu-oxo bridge. ENDOR shows that the major conformer has a histidine and a water bound to both Fe ions. In the minor conformer, one of the irons has lost the terminal water ligand. The structure of the trapped [DS(ox)](mv) state relaxes upon annealing to 170 K: the mu-oxo bridge in the major cryoreduced DS species protonates on annealing to 170 K; this does not occur for the major DS-substrate complex, even upon annealing to 230 K. The relaxed Fe(II)Fe(III) center in cryoreduced DS and DS-substrate show much less intense and resolved (14)N ENDOR spectra than those of the structurally similar cryoreduced diiron center in ribonucleotide reductase (RNR) protein R2. This difference may reflect some differences in His-Fe bonds. The alterations in the diferric site of DS induced by substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the diferric site. The mixed-valence states of DS are unstable at temperatures above 230 K.

tRNA-modifying MiaE protein fromSalmonella typhimuriumis a nonheme diiron monooxygenase

“…3B) could be attributed to a double quanta-double quanta correlation from another strongly coupled nitrogen with ͉ a N ͉ equal to 4.1 MHz, indicative of the presence of a second nitrogen ligand. Observation of hyperfine coupling of two nitrogens from histidine ligands in similar systems was reported previously (31). Hyperfine values obtained here are compatible with those obtained for the [Fe II -Fe III ] clusters of the cryoreduced R2 protein (3.16 and 7.31 MHz) and of the cryoreduced stearoyl carrier ⌬ 9 -desaturase protein (3.34 and 9.1 MHz) (31).…”

“…Observation of hyperfine coupling of two nitrogens from histidine ligands in similar systems was reported previously (31). Hyperfine values obtained here are compatible with those obtained for the [Fe II -Fe III ] clusters of the cryoreduced R2 protein (3.16 and 7.31 MHz) and of the cryoreduced stearoyl carrier ⌬ 9 -desaturase protein (3.34 and 9.1 MHz) (31). Similar values were obtained for both the His-tagged and untagged forms of MiaE2.…”

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

“…In both the soluble and membrane desaturases, electrons are supplied singly by interaction of the desaturase with ferredoxin and cytochrome b 5 , respectively. In vitro redox titration of the soluble desaturase at ambient temperatures failed to reveal the presence of a meta (Fe 2ϩ -Fe 3ϩ ) state of the iron center that is observed upon reduction at low temperatures (77 K), suggesting that the meta state is unstable at ambient temperatures (31). A dimeric structure of the desaturase would potentially allow for concerted reduction of the iron centers within each protomer to produce two meta state iron centers.…”

Cloning and Characterization of a Stearoyl–Acyl Carrier Protein Desaturase Gene from Cinnamomum longepaniculatum

“…SAD plays a key role in determining the ratio of saturated fatty acids to unsaturated fatty acids in plants (Lindqvist et al 1996), and this ratio is closely associated with many functions in plants, particularly of plants acclimation to low temperatures (Kodama et al 1995). Many SAD genes have been cloned from different plants, and the structures and functions of several SAD have been studied (Davydov et al 2005;Lindqvist et al 1996). Antisense expression of the Brassica rapa SAD gene in Brassica nupus led to dramatically increased stearate levels (up to 40%) in seeds of transgenic B. nupus (Knutzon et al 1992).…”