Conclusions

By treating a larger part of the protein by QM methods, a higher basis set and a crystal struc-ture of higher resolution, absolute reduction potentials forAnabaenaFdx could be calculated, which are in much better agreement with measured values than previous results. Also for the first time the asymmetric environment of the two iron atoms was included sufficiently into calculations leading to two energetically well separated reduced forms. The results are in agreement with the M¨ossbauer data on a localized mixed valence F e²⁺/F e³⁺ state. Also the reduced iron Fe1 is in agreement with NMR data. However, obtaining accurate energies by QM calculations still remains a challenge. It seems that todays DFT functionals are not accurate enough for the purpose of this work. An additional problem is to derive accurate point charges form the electron density calculated by the QM method. This problem has no unique solu-tion and different approaches find considerably different results. Certainly, the electrostatic calculations critically depend on the set of point charges used.

Another problem is that reduction potentials are usually measured at pH 7, but no crystal structure exists at this pH. The used crystal structure was determined at a pH of 5.5, at which acidic groups are likely to be protonated (especially in such an acidic protein as Fdx and with a negatively charge iron-sulfur center). Under such conditions, groups of acidic residues arrange into orientations stabilizing a bound proton and by that leading to an in-creasedpKa value compared to the groups separated in solution. Similarly, Glu94 adopts an orientation close to the negatively charged iron-sulfur cluster stabilizing its protonated form.

Probably the residue would adopt a different orientation at higherpH. It seems that suchpH dependent conformational changes are important in Fdx since a structure could be modeled,

which stabilizes a deprotonated Glu94 at neutral pH leading to very good agreement with experimental reduction potentials.

Due to the largely over-estimated favorable energy for the NH-in conformer by the QM method, it can only be speculated about the potential role of the conformational change in the peptide bond. The interpretation of Moraleset al., that the CO-in conformer is only populated in the oxidized form and the NH-in conformer is only populated in the reduced form seems unlikely at present. The calculations on the CO-in conformer show that a reduction potential close to experiment can be obtained for this conformation only. The energy difference between the CO-in and the NH-in conformer is currently by far too large to obtain a reduction potential in the order of magnitude of the measured values. Shifting the energy of the NH-in conformer by about 10 ^kcal_mol, would lead to results only showing the CO-in conformer in the oxidized form in agreement with experiment. The oxidized form of the NH-in conformer would be 2-3 ^kcal_mol higher in energy and therefore not be significantly populated. For a lower energy shift, the CO-in and NH-in conformer would become equi-energetic and should have both been observed in the oxidized form or for energy shifts lower than,e.g.,5 ^kcal_mol only the NH-in conformer should have been observed in the oxidized form. For an assumed shift of 10 ^kcal_mol, the energy of the reduced NH-in conformer is about 1 ^kcal_mol lower than the CO-in conformer. By that, both conformers would be populated as the crystallographic data on the reduced crystal could be interpreted. For higher energy shift values, again only the CO-in conformer would be populated also in the reduced form. By this thermodynamic interpretation, in which the two conformers are about equi-energetic in the reduced form and only the CO-in conformer is populated in the oxidized form, a direct role of the conformational change for the reduction potential could be ruled out. However, the conformational change may still influence the kinetics of electron transfer by lowering the barrier. The NH-in conformation seems to provide a more direct route for transferring electrons to FNR.

5.4 Protonation Probability Calculations of Cu

Ligands in the Reaction Mechanism of Cytochrome c Oxidase

The reaction mechanism of Cytochrome c oxidase (CcO) is studied by Punnagai Munusami in our group. The complex protonation equilibria in the active site are studied using Perl MoleculeandQMPB. Our work is described in [60].

Cytochromec oxidase is the terminal enzyme in the respiratory chain. It reduces oxygen to water by consuming four protons. The oxygen reduction drives pumping of four additional protons across the membrane [193–195]. However, details of the reaction mechanism are not well understood and under debate [58, 59, 196–198].

The enzyme contains several metal prosthetic sites and 13 subunits in mammals. The CuA

center accepts electrons from Cytochromec and transfers them to hemea and finally to the heme a3 - CuB binuclear center, where oxygen gets reduced. The CuB center consists of a copper ion coordinated by three histidines. At the fourth position the oxygen, which gets reduced to water, is coordinated (Fig. 5.13). One of the coordinating histidines, His240, is cross-linked with Tyr244.