Journal
BIOCHEMISTRY
Volume 45, Issue 15, Pages 4848-4858Publisher
AMER CHEMICAL SOC
DOI: 10.1021/bi0600547
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Funding
- NIGMS NIH HHS [R01 GM050853, R01 GM050853-13, GM-50853, T32 GM-08293] Funding Source: Medline
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Stearoyl-acyl carrier protein desaturase (Delta 9D) catalyzes the 02 and 2e(-) dependent desaturation of stearoyl-acyl carrier protein (18:0-ACP) to yield oleoyl-ACP (18:1-ACP). The 2e- are provided by essential interactions with reduced plant-type [2Fe-2S] ferredoxin (Fd). We have investigated the protein-protein interface involved in the Fd-Delta 9D complex by the use of chemical cross-linking, site-directed mutagenesis, steady-state kinetic approaches, and molecular docking studies. The treatment of the different proteins with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide revealed that carboxylate residues from Fd and lysine residues from Delta 9D contribute to cross-linking. The single substitutions of K60A, K56A, and K230A on Delta 9D decreased the k(cat)/K-M for Fd by 4-, 22-, and 2400-K60A respectively, as compared to wt Delta 9D and a K41A substitution. The double substitution K56A/K60A decreased the k(cat)/K-M for Fd by 250-fold, whereas the triple mutation K56A/K60A/K230A decreased the k(cat)/K-M for Fd by at least 700 000-fold. These results strongly implicate the triad of K56, K60, and K230 of Delta 9D in the formation of a catalytic complex with Fd. Molecular docking studies indicate that electrostatic interactions between K56 and K60 and the carboxylate groups on Fd may situate the [2Fe-2S] cluster of Fd closer to W62, a surface residue that is structurally conserved in both ribonucleotide reductase and mycobacterial putative acyl-ACP desaturase DesA2. Owing to the considerably larger effects on catalysis, K230 appears to have other contributions to catalysis arising from its positioning in helix 7 and its close spatial location to the diiron center ligands E229 and H232. These results are considered in the light of the presently available models for Fd-mediated electron transfer in Delta 9D and other protein-protein complexes.
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