The 2-His-1-carboxylate facial triad is a ubiquitous active site structural motif in oxygen activating mononuclear non-heme iron enzymes. The α-KG-dependent halogenases are structurally different, however, having a Cl- ligand instead of the facial triad carboxylate. In the present study, CD, MCD, and VTVH MCD spectroscopies have provided insight into the active site geometric and electronic structures of the α-KG-dependent halogenase CytC3. From a comparison to data on CS2 and parallel studies of TauD, these results provide insight into the role of the facial triad in the α-KG-dependent oxygenases.
A commonly considered role of the facial triad in oxygen activating mononuclear non-heme iron enzymes is to provide three amino acid ligands at the enzyme active site to tightly bind FeII
. By near-infrared CD, the effect of loss of the carboxylate of the facial triad on FeII
binding to the active site of CytC3 was evaluated. No CD signal due to FeII
binding was observed, giving an estimate for the upper limit for the binding constant of FeII
to apo-CytC3 in the presence of Cl-
that is at least two orders of magnitude lower than that for CS2. These results demonstrate the requirement of the facial triad for providing a site for high-affinity FeII
binding. In CytC3, the lack of FeII
binding with only two amino acid ligands is overcome by the addition of the α-KG co-substrate. Crystallography shows that the α-KG cofactor binds to the active site even in the absence of FeII
and supplies two additional ligands to create a high affinity iron binding site.21
Importantly, MCD studies of CytC3/FeII/Cl-/α-KG show the presence of a 6C ferrous site with a large 5Eg splitting (Δ5Eg ~ 3460 cm-1), indicating the presence of a weakly coordinated water ligand (, blue). This assignment is based upon LF calculations which indicate that the Cl- ligand (at ~2.44 Å from crystallography on the related enzyme SyrB2) would not lead to the large 5Eg splitting observed. An analogously large 5Eg splitting (Δ5Eg ~ 3260 cm-1) is also observed in TauD/FeII/α-KG and must be due to the presence of a weak water ligand. These results contrast with our previous MCD data for CS2/FeII/α-KG, in which the 5Eg splitting (Δ5Eg ~ 1630 cm-1) was consistent with a strong water ligand at the sixth coordination position (, red). Given that the resting site of TauD is 6C with water tightly bound to FeII from MCD, we conclude that it is the bidentate coordination of α-KG cofactor, a good donor (which replaces two water ligands in the resting site), that leads to the weakening of the remaining water ligand in the α-KG complexes.
The low-temperature, 7T MCD spectra of CS2/FeII/α-KG (red), TauD/FeII/α-KG (green) and CytC3/FeII/Cl-/α-KG.
Alhough α-KG binding in CS2 could also lead to the weakening of the water ligand, this weakening is not observed, as MCD data show that the water is bound tightly to FeII
even in the presence of α-KG. An active site feature that can stabilize water binding in CS2 is a H-bonding interaction between the coordinated water and the non-coordinating oxygen of the monodentate carboxylate of the facial triad (observed in the crystal structure of CS2/FeII
). Replacement of this carboxylate ligand in CytC3 by Cl-
eliminates this H-bonding interaction, which could lead to weakening of the water ligand upon α-KG binding. While TauD contains this carboxylate ligand it appears to be in an orientation (i.e. potentially flipped down as observed in TauD crystallography) that would preclude a stabilizing H-bond with the coordinated water, leading to its weakened ligation. Thus, a role of the facial triad in oxygen activating mononuclear non-heme iron enzymes appears to be the regulation of water affinity to the site upon α-KG binding through H-bonding to the non-coordinating oxygen of the carboxylate ligand.
Density functional theory (DFT) calculations were utilized to gain further insight into the contribution of this H-bonding interaction to the water binding to the ferrous site ().d
In order to estimate the strength of the H-bond, two structures were optimized – one with the carboxylate O in the plane of the α-KG cofactor and the other with the carboxylate rotated to allow for H-bonding to the water ligand. The difference in energy of the two structures was found to be ~8.6 kcal/mol, giving an upper estimate of the strength of the H-bond. This strong H-bond is due to the anionic nature of the carboxylate, which also polarizes the O-H bond of the water ligand, leading to more OH-
character and a shorter Fe-O bond. This H-bonding to the water decreases its binding energy to the 5C FeII
site from ΔG = +8.0 kcal/mol (calculated without the H-bond, , left) to ΔG = -1.0 kcal/mol with the H-bond (, right). Thus, the presence of the H-bond to the terminal oxygen of the carboxylate of the facial triad significantly increases the water affinity of the FeII
Geometry optimized structures of TauD/FeII/α-KG without and with H-bonding to carboxylate.
In α-KG-dependent oxygenases, the presence of a 6C α-KG bound complex is important in preventing an uncoupled reaction in the absence of substrate. From the above results, these enzymes generally maintain the 6C site through H-bonding between the carboxylate ligand and the coordinated water (as for CS2). TauD and CytC3 do not, having a weak water ligand in their α-KG complexes which appears to be sufficient to prevent the uncoupled reaction with oxygen.e
TauD and CytC3 appear to employ additional second sphere interactions to prevent complete loss of water ligation upon α-KG binding, which would generate a 5C site highly susceptible to uncoupled O2
activation. From crystallography, an Asn residue is present in TauD near the water coordination position that could potentially H-bond to the water to stabilize weak ligation. This residue is absent in the available crystal structures of other α-KG-dependent enzymes. In CytC3, a second sphere H-bonding interaction involving the coordinated Cl-
and the water ligand via a non-coordinated water could serve the same stabilizing function (according to the structure of the homologous SyrB2).21
In addition to defining a role of the carboxylate of the facial triad, CD and MCD studies of substrate binding in CytC3 and TauD provide insight into O2
activation and the reaction pathways of these enzymes. In both systems, substrate binding leads to similar ES complexes with α-KG bound in a bidentate fashion. The presence of the Cl-
ligand in the first coordination sphere of CytC3 has little direct effect on the electronic structure of the substrate-bound complex. The observed 6C→5C conversion of the FeII
sites upon substrate binding to the α-KG complexes provides an open site for O2
to bind and react, following a common mechanism for O2
activating mononuclear non-heme iron enzymes which utilize redox active cofactors. Upon O2
binding to the coordinatively unsaturated FeII
site in the presence of both cosubstrates, these enzymes would proceed along similar reaction coordinates with decarboxylation of the coordinated α-KG leading to a similar iron-oxygen intermediate as has been demonstrated.15, 22
It is interesting to consider the possible mechanistic role of the weakened water ligands in CytC3 and TauD compared to CS2 in the 6C→5C conversion of the FeII
site upon substrate binding required for O2
activation. From crystallography, the substrate in CS2 binds directly above the water coordination position, leading to the loss of the water ligand to generate the 5C site.12
In contrast, from crystallography of TauD, substrate (taurine) does not bind directly above the water coordination site but is more off-center.13, 36
Thus, the bound taurine would likely interact more weakly with the coordinated water ligand and, hence, the weakened water ligand in TauD/FeII
/α-KG would facilitate its loss of water upon taurine binding. The presence of a weak water ligand in CytC3 may also be required for the 6C→5C conversion. The decreased fraction of 5C complex generated in CytC3 upon substrate binding (compared to TauD) indicates that even with a weak water ligand, l
-Aba-S-CytC2 substrate binding is not as efficient in destabilizing the water ligand. One possible explanation is that the CytC3 substrate does not approach as close to the FeII
site as observed in other α-KG-dependent oxygenases, possibly a consequence of its attachment to a carrier protein.
Finally, it is interesting to consider the active site features that could contribute to the observed chlorination rather than hydroxylation reactivity of CytC3. After H-atom abstraction by the FeIV
=O intermediate generated in the O2
reaction, the presence of the Cl-
ligand in the first coordination sphere of the resulting FeIII
species in CytC3 might allow the site to transfer either a hydroxyl radical (as in TauD) or a chlorine atom to the substrate radical. From our MCD studies, the substrate in CytC3 is likely fairly distant from the iron site, consistent with the reduced rate of C-H bond cleavage for this enzyme.22
In this case, during the rebound reaction the FeIII
-L (L = OH-
) bond would largely be broken prior to formation of the Csub
-L bond. Therefore, the relative reaction barriers for rebound hydroxylation versus chlorine atom transfer would depend upon the energetic differences between the homolytic cleavage of an FeIII
bond or an FeIII
bond. As raised in ref. 45
, there is a difference in potential and, in addition, a difference in bond dissociation energy (C-Cl < C-OH by ~8-11 kcal/mol in aliphatic and aromatic compounds46
), both of which will favor chlorination. It is also noteworthy that because the chlorine atom transfer would result in reduction of the FeIII
species, protonation of the hydroxide ligand to generate an FeII
site would be strongly coupled to and potentially help promote the chlorine transfer.
In summary, CD and MCD spectroscopies have been utilized to elucidate the geometric and electronic structure of the α-KG-dependent halogenase CytC3, which has an unusual FeII coordination site in which Cl- replaces the carboxylate of the 2-His-1-carboxylate facial triad. This perturbation eliminates FeII binding to apo-CytC3, supporting the necessity of the facial triad for iron coordination to form the resting site and the role of the α-KG in the chlorinase in forming the catalytic site. Interesting differences in the α-KG complex indicate the presence of a weak water ligand. Combined with parallel studies of TauD and past studies of CS2, these results define a role of the carboxylate ligand of the facial triad in stabilizing water coordination via a H-bonding interaction between the non-coordinating oxygen of the carboxylate and the coordinated water. Finally, these studies provide initial insight into the active site features that favor chlorination versus hydroxylation in CytC3.