Spectroscopic Characterization of Soybean Lipoxygenase-1 Mutants:  the Role of Second Coordination Sphere Residues in the Regulation of Enzyme Activity

Abstract

Lipoxygenases are non-heme iron enzymes, which catalyze the stereo-and regiospecific hydroperoxidation of unsaturated fatty acids. Spectroscopic studies on soybean lipoxygenase have shown that the ferrous form of the enzyme is a mixture of five-and six-coordinate species (40 and 60%, respectively). Addition of substrate leads to a purely six-coordinate form. A series of mutations in the second coordination sphere (Q697E, Q697N, Q495A, and Q495E) were generated, and the structures of the mutants were solved by crystallography [Tomchick et al. (2001) Biochemistry 40, 7509-7517]. While this study clearly showed the contribution of H-bond interactions between the first and the second coordination spheres in catalysis, no correlation with the coordination environment of the Fe II was observed. A recent study using density-functional theory [Lehnert and Solomon (2002) J. Biol. Inorg. Chem. 8, 294-305] indicated that coordination flexibility, involving the Asn694 ligand, is regulated via H-bond interactions. In this paper, we investigate the solution structures of the second coordination sphere mutants using CD and MCD spectroscopy since these techniques are more sensitive indicators of the first coordination sphere ligation of Fe II systems. Our data demonstrate that the iron coordination environment directly relates to activity, with the mutations that have the ability to form a five-coordinate/six-coordinate mixture being more active. We propose that the H-bond between the weak Asn694 ligand and the Gln697 plays a key role in the modulation of the coordination flexibility of Asn694, and thus, is crucial for the regulation of enzyme reactivity.Lipoxygenases (LOs) 1 are mononuclear non-heme iron dioxygenases that catalyze the stereo-and regiospecific hydroperoxidations of cis,cis-1,4 polyunsaturated pentadienyl containing fatty acids. LOs have been isolated and characterized from numerous mammalian and plant sources and are believed to occur ubiquitously throughout these two phyla (1-3). In mammalian organisms, there are multiple LOs with differing regio-and stereospecificity against their substrate, arachidonic acid (AA). The products of these reactions are further metabolized by an array of enzymes leading to the formation of two major classes of cell effector molecules, leukotrienes and lipoxins, which are mediators in anaphylactic and inflammatory disorders (4, 5). Additionally, LO products are involved in a variety of metabolic functions, including organelle degradation (6), transcription regulation (7), and possibly tumor cell metastasis (8). For plant LOs, the main substrates are linoleic acid (LA) and linolenic acid, which are further metabolized to plant hormones, such as jasmonic acid. These hormones are involved in a variety of metabolic functions including plant growth and development, senescence, and cellular response to wounding and infections by pathogens (1). Soybeans have three LO isoforms (sLO-1, sLO-2, and sLO-3), which have homologous sequences and molecular weights of ∼96 kDa (9). This...

Enzyme structure and dynamics affect hydrogen tunneling: The impact of a remote side chain (I553) in soybean lipoxygenase-1

“…4 and 5) raised the issue of whether this structural change could also be impacting the experimental rate parameters. We consider this explanation highly unlikely for a number of reasons that include: (i) the observation of two configurations for the H499/Q495 hydrogen bonding pair in WT-SLO; (ii) the fact that the full H-bonding network is retained, along with the liganding of H499 to the iron, among all of the mutants; and (iii) the published finding that the Q495E retained full enzyme activity (28).…”

Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX)

“…Three residues of the second-coordination sphere (35) around the iron coordination sphere, Q784 Q711,Q697 and L842…”

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

“…The signals in the EPR spectra and their intensity are a reflection of the detailed interactions of iron and its ligands, and the two signals may correspond to 5-and 6-coordinate iron liganding. Previously, a network of H-bonds involving iron ligand Asn 694 and residues in the iron second coordination sphere has been characterized by mutation, CD, magnetic circular dichroism, and x-ray structure analyses, with the conclusion that dynamic changes in iron coordination, and especially in the iron-Asn 694 bond length, is essential for catalytic function (44,45). It is likely that the helix 11 containing Ala or Gly at 542 is connected through hydrogen bonds of residues and waters in cavity IIa to the network of H-bonds that modulate the iron-ligand bond lengths.…”