A first sphere mutant of sLO-1 (N694C) has been prepared in which the Asn694 ligand responsible for the coordination flexibility observed in WT sLO-1 has been selectively mutated to a Cys. A combination of CD and VTVH MCD spectroscopies indicates that the FeII site of N694C is a 5C square pyramidal species in contrast to the 40/60 % mixture of 5C and 6C ferrous species in WT sLO-1/FeII. The Cys residue is not coordinated to the FeII site of the mutant, but oxidation of the site leads to ligation of the cysteine sulfur to the FeIII based upon the presence of a Cys-FeIII charge transfer band at 762 nm (ε = 760 M-1 cm-1).
The observed Cys-FeIII
charge transfer band in N694C is at lower energy with decreased intensity compared to the Cys-FeIII
charge transfer transition in ferric high-spin sites such as superoxide reductase.33, 34
One possible contribution to the decreased intensity of the transition could be partial coordination of the ferric site by Cys. However, from EPR of ferric N694C the rhombic signal, indicative of a 6C site in sLO where the Cys S occupies the sixth coordination site, is present at 80%. Correcting for this still leads to a factor of ~2.5 lower extinction coefficient relative to superoxide reductase (ε(SOR at 660 nm) = 2500 M-1
However, a weakened FeIII
-S bond in N694C compared to superoxide reductase would both decrease the intensity of the transition and shift it to lower energy as observed. A weaker Cys-FeIII
bond could reflect the fact that the cysteine side chain is one carbon shorter than the Asn ligand it replaced. This is also consistent with the loss of the Cys ligand upon reduction of the site.
Previous studies of WT sLO-1 have shown that it is converted from a 6C FeII
site upon substrate analog binding to a 5C FeIII
In contrast, the present study indicates that N694C sLO-1 is converted from a 5C FeII
site with a substrate analog bound (oleic acid, see S.I.) to a 6C FeIII
site as determined from EPR data which show a predominately rhombic site indicative of 6C.37
form of N694C is the catalytically active species analogous to WT sLO-1 as determined by UV/Vis absorption and EPR studies of the decay of this species at a catalytically relevant rate in the anaerobic reaction with substrate. Importantly, the kcat
for N694C is significantly reduced (~ 3000-fold) relative to WT sLO-1 and the iron active site in the mutant is anaerobically reduced upon substrate addition.
Our spectroscopic studies of N694C support the presence of an iron active site for N694C with a lowered Eo of the FeII/FeIII couple compared to WT sLO-1 due to the presence of a 6C ferric site, with the cysteine sulfur coordinated in the ferric but not ferrous state. This stabilizes the FeIII site relative to the FeII site. The pKa of the coordinated water in the 6C ferric site of the mutant should be increased as the site is 6C and the cysteine donor ligand would tend to decrease the donation of coordinated H2O to FeIII. Thus, the Asn694→Cys694 mutation results in an iron active site with an Eo of the FeII/FeIII couple and a pKa of water bound to the ferric site that would, in principle, be more effective than WT sLO-1 in a σ-organoiron mechanism. Alternatively, with respect to H-atom abstraction, the coordination change relative to WT sLO-1 (loss of the Cys ligand to produce a 5C FeII site) would both lower Eo of the FeII/FeIII couple and lower the pKa of water bound to the ferrous site which would disfavor this reaction. In fact, the enzymatic turnover data are consistent with an H-atom abstraction mechanism for N694C due to the observation of a significant deuterium isotope effect on rate, a substantially reduced kcat (~ 3000-fold) of N694C relative to WT sLO-1 and the observed anaerobic reduction of iron by substrate. The iron active site would remain in the ferric state throughout the catalytic cycle in a σ-organoiron mechanism.
In addition to Eo
, possible structural changes due to the Asn694→Cys694 mutation could contribute to the decreased reactivity. Since the cysteine sulfur is not coordinated in N694C/FeII
, the ligands likely remain unchanged relative to the WT sLO-1/FeII
5C component (3His, 1 C-terminal carboxylate from Ile839 and H2
O). However, a perturbation of the 5C ferrous site of N694C is observed compared to the 5C site in WT sLO-1/FeII
() in that both excited state transitions have shifted to higher energy and the sign of the higher energy transition has become negative.12
In addition, the splitting of the 5
ground-state obtained from the VTVH MCD data in (inset) has decreased in the mutant. Previously it has been shown that the N694 ligand is involved in a H-bonding network with residues Q697 and Q495 in the second sphere, linking the substrate binding pocket to the flexible N694 ligand and, thus, playing a role in the coordination flexibility of this residue ().11
Binding of substrate, glycerol or inhibitor (oleic acid) affects the H-bonding, resulting in conversion to a purely 6C site.20
However, the N694C mutation eliminates the possibility of a H-bond from residue 694 to Q697, supported by CD studies showing no perturbations of the FeII
due to glycerol or substrate analog oleic acid binding (see Figure S3
). Crystallographic studies of other first sphere mutants (N694H and N694G) 11, 24
which also lack H-bonding from residue 694 to Q697 indicate that perturbing this network also can affect the interactions of these second sphere residues with the coordinating H499 ligand and, thus, its interaction with the FeII
. Therefore, the changes in the MCD spectrum of N694C/FeII
relative to WT sLO-1/FeII
are consistent with the perturbation of the H-bonding network and the effects of this on the H499 ligand. However, since both N694C/FeII
and WT sLO-1/FeII
are 5C with the same ligation, this structural perturbation should not have a significant effect on Eo
. For the catalytically relevant ferric site of N694C sLO-1, the structural difference from WT sLO-1/FeIII
is the ligation of Cys which could sterically affect substrate binding. However, the fact that the KM
value for the N694C mutant is similar to that for WT sLO-1 and to that previously reported for another first sphere mutant, N694H (KM
~ 4 μM),23
indicates that the mutation results in only minor changes in the substrate binding pocket.
Active Site of WT sLO-1 including the second sphere H-bonding network.
Finally, based on crystallographic studies of WT sLO-1 in the absence of substrate, it was proposed that the presence of certain active site residues such as Gln495 and Gln697 restrict access to the Fe site to disfavor formation of the σ-organoiron intermediate.10
However, the possibility of structural rearrangement of the protein active site to accommodate this species could not be eliminated. Subsequent crystallographic studies do indicate the possibility of structural changes in some of these residues upon substrate binding.11
However, the spectroscopic and kinetic data presented here argue against a σ-organoiron intermediate.
In summary, a first sphere mutant of sLO-1, N694C, has been prepared and characterized which allows evaluation of the σ-organoiron mechanism previously proposed for the reaction of lipoxygenases. While the mutant has a lowered Eo of the FeII/FeIII couple and a raised pKa of water bound to the ferric site which would favor a σ-organoiron reaction, the kinetic data of N694C show a significant deuterium isotope effect on rate, an observed anaerobic reduction of iron by substrate and a substantially reduced kcat (~ 3000-fold) of N694C relative to WT sLO-1. These results experimentally evaluate the organoiron mechanism and provide further support for H-atom abstraction as the catalytically relevant mechanism in lipoxygenase.