Geometry and Electronic Structure of the P-Cluster in Nitrogenase Studied by Combined Quantum Mechanical and Molecular Mechanical Calculations and Quantum Refinement

We have studied the geometry and electronic structure of the P-cluster in nitrogenase in four oxidation states: P N , P 1+ , P 2+ , and P 3+ . We have employed combined quantum mechanical and molecular mechanical (QM/MM) calculations, using two different density-functional theory methods, TPSS and B3LYP. The calculations confirm that the side chain of Ser-188 is most likely deprotonated in the partly oxidized P 1+ state, thereby forming a bond to Fe6. Likewise, the backbone amide group of Cys-88 is deprotonated in the doubly oxidized P 2+ state, forming a bond to Fe5. The calculations also confirm the two conformations of the P-cluster in the atomic-resolution crystal structure of the enzyme, representing the P N and P 2+ states, but show that the finer differences between the two structures are not fully reflected in the crystal structure, because the coordinates of only two atoms differ between the two conformations. However, the recent crystal structure of the P 1+ state seems to be of lower quality with many dubious Fe-Fe and Fe-S distances. Quantum refinement of this structure indicates that it is a mixture of the P 1+ and P 2+ states but confirms that the side chain of Ser-188 is most likely deprotonated in both states. TPSS gives structures that are appreciably closer to the crystal structures than does B3LYP. In addition, we have studied all 16-48 possible broken-symmetry states of the four oxidation states of the P-cluster with DFT in the one or two observed spin states. For the reduced P N state, we can settle the most likely state from the calculated energies and geometries. However, for the more oxidized states there are large differences in the predictions obtained with the two DFT methods.