We will compare some aspects of the reaction pathway and properties of the O2 carrier protein hemerythrin (Hr) with the early parts of the reaction cycles of the structurally related enzymes methane monooxygenase hydroxylase (MMOH) and ribonucleotide reductase (RNR). A general structural comparison of these three proteins is given inFigure 4 for the diferric and diferrous Figure 3 Schematic partitioning of space and assignment of different dielectric regions. (a) Quantum region vs. continuum solvent; (b) three region model: active site (quantum region); protein, and solvent;
(c) dielectric model for (b) showing generating charges for reaction field potential from the quantum cluster (d) same dielectric model showing protein generating charges for the protein field potential.
forms.53–55Hemerythrin functions as a reversible carrier for molecular oxygen, while MMOH and RNR undergo redox-dependent cluster activations which then prepare the active sites for sub- sequent reactions with molecular oxygen and then with substrates by producing high-valent iron- oxo (or oxo, hydroxo) intermediates. MMOH hydroxylates methane to methanol, and can perform hydroxylation and other quite diverse transformations on a variety of hydrocarbons.
The iron-oxo dimer site of RNR generates a stable tyrosine radical, which then serves as a catalytic radical transfer site for reduction of ribonucleotides to deoxyribonucleotides.
In contrast to MMOH and RNR, Hr has the diferrous form as the ‘‘resting unreacted’’ state, and there is a single five-coordinate site for end-on binding of O2. After O2binding, a Fe end-on bound hydroperoxide (OOH)intermediate is formed, with the iron centers becoming diferric. In Hr, the O2 binding step occurs comparatively earlier, and the oxidation states of the first two intermediates are reversed compared to RNR and MMO. The resting states of both are diferric
Fe O H
Fe H O
O O
O O His246 His147
O OH2
Glu144
O Glu209 O
Glu243 Glu114 O
Fe O Fe
O O
O O His246 His147
O OH2
Glu144
O Glu209 Glu114 O
Glu243
O Fe H2O
O O
O O His241 His118
O
Glu115
O Glu204 O
Glu238
O Asp84
Fe O
Fe
O O
O O
His241 His118
O
Glu115
O Glu204 Asp84 O
Glu238
Fe OH2
Fe O
Fe
O O
O His73
His77
His54
Glu58
His101 His25
Asp106
deoxyHr
MMOHox MMOHred
RNRox RNRred
OH2
H Fe O
O
Fe
O O
O His73
His77 His54
Glu58
His101 His25
Asp106
oxyHr O O OH
FeIIFeII FeIIIFeIII
Figure 4 Active site structures and corresponding oxidation states for iron-oxo dimer proteins: methane monooxygenase (MMOH); ribonucleotide reductase (RNR); and hemerythrin (Hr).
502 Density Functional Theory
(MMOHox, RNRox) and the interaction with reductases allows the formation of the diferrous forms after net two-electron transfer and coupled protonation. These reactions open up the active sites of MMOH and RNR for initial bidentate coordination to 2Fe and reaction with molecular oxygen driving the reaction further into the catalytic cycle. As can be seen by comparing the diferrous and diferric structures of RNR and MMO, along with computational analysis of likely reaction steps along these paths, the activation of the 2Fe sites involves both electronic charge transfer and protonation of the bridging (OH) in MMO, and-O in RNR with the pathway facilitated by protonation of a nearby monodentate glutamate residue. This is the same Glu that undergoes a carboxylate shift to 1,1 OCO bidentate in MMOH and 1,3 OCO bidentate in RNR.
Based on Brunold and Solomon’s combined DFT and spectroscopic work,54,55 the reaction pathway of Hr can be followed in some detail (seeFigure 5). The deoxyHr form has a resting diferrous oxidation state that can donate electron density to the O2as it binds. The single bridging OH of deoxyHr transfers its proton to stabilize the developing end-on peroxide from O2; the bound hydroperoxide can still form a hydrogen bond to the remaining bridging oxo of the FeIIIOFeIII unit. The high covalency of the bridging diferric-oxo unit allows for a stable intermediate. In the diferric oxyHr, the stronger AF coupling compared to diferrous deoxyHr provides part of the driving force for O2 binding, an effect estimated as about 6.6 kcal mol1 comparing the high-spin with the Sẳ0state of oxyHr. This is based on the experimental J coupling, which is in reasonable qualitative agreement with the calculated J coupling for a simplified model. Considering the O2dissociation process, the presence of a very weakly coupled manifold of deoxyHr spin states with Stotẳ0–4 and the product tripletO2(3g) (formed from the bondedO2(1g state) allows for a more pronounced barrier with a crossover between the Stotẳ0state of oxyHr and the family of deoxyHr spin states.
The protonation states of these systems play important roles in the chemical transformations along the reaction pathways, but these are difficult to examine directly via X-ray crystallography in the absence of very high-resolution structures. Even here, only some states of a reaction cycle may be available. It is therefore valuable to examine theoretical calculations both of active site geometries and of HeisenbergJcouplings, which reflect the coordinating ligands, the protonation state of bridging (OHn) groups, and the Fe site oxidation states. Evalution of pKas by combined DFT and electrostatics methods further clarifies the picture of protonation states for MMOH.
These computational results can be compared with spectroscopic observations and X-ray struc- tures of synthetic model systems as well as protein structures.
Table 1shows how Heisenberg J parameters calculated for different Fe oxidation states and bridges compare with experiment. The diferric-oxo AF couplings are much stronger than
0 5 10 15 20 25 30 35 40
Energy (kcal mol)–1
I III
oxyHr (1 )
deoxyHr(5 )
Fe2 Fe1
O Oa
Ob H
Fe2 Fe1
O Oa
Ob H
Fe2 Fe1
O Oa
Ob H
Fe2 Fe1
O Oa
Ob H
Fe2 Fe1
O Oa
Ob
H +
(2 )
(4 ) (3 )
1
2
3
4
5
S=Ms=5 Ms=0
∞ r(Fe -O )2 a r(O -H)b r(Fe -O )2 a
II
2.0 2.2 2.4 2.6 2.8 1.2 1.4 1.6 3.0
Figure 5 Reaction pathway of oxygen binding to hemerythrin. Adapted and simplified from Brunold and Solomon (1999).54,55
diferric-hydroxo type, while the diferrous couplings are generally weaker still. The protonation state of the bridging oxygen ligands is also strongly reflected in the iron-bridging oxygen bond lengths as we have demonstrated in Lovell et al.52,53,56 Based on evaluation of protonation energetics and tautomeric states, as well as orbital and structural analysis, we proposed the carboxylate shift mechanism in Figure 6 for the proton coupled two-electron redox process which gives the diferrous MMOH(red) state. The proton is carried by Glu243 as it undergoes the carboxylate shift. The energetics of the subsequent steps of the reaction cycle have been considered by a number of groups. It is important to assess both structures and spectroscopic properties of predicted intermediates by comparison of calculated structures and properties with experiment.57 These comparisons will further test the intermediates and related transition states that have been proposed based on calculated DFT energies.5,58–60