Structure refinement of the aldehyde oxidoreductase from Desulfovibrio gigas (MOP) at 1.28 angstrom

The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio g igas (MOP) is a member of the xanthine oxidase family of enzymes. It has 90 7 residues on a single polypeptide chain, a molybdopterin cytosine dinucleo tide (MCD) cofactor and two [2Fe-2S] iron-sulfur clusters. Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crysta ls of this enzyme in the oxidized form. The cell constants of a = b = 141.7 8 and c = 160.87, Angstrom are about 2% shorter than those of room temperat ure data, yielding 233,755 unique reflections in space group P6(1)22, at 1. 28 Angstrom resolution. Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and R-fr ee factor of 19.3 (4 sigma cut-off) with "riding" H-atoms at their calculat ed positions. The model contains 8146 non-hydrogen atoms described by aniso tropic displacement parameters with an observations/parameters ratio of 4.4 . It includes alternate conformations for 17 amino acid residues. At 1.28 r esolution, three Cl and two Mg2+ ions from the crystallization solution wer e clearly identified. With the exception of one Cl- which is buried and 8 A ngstrom distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing. The overall structure has n ot changed in comparison to the lower resolution model apart from local cor rections that included some loop adjustments and alternate side-chain confo rmations. Based on the estimated errors of bond distances obtained by block ed least-squares matrix inversion, a more detailed analysis of the three re dox centres was possible. For the MCD cofactor, the resulting geometric par ameters confirmed its reduction state as a tetrahydropterin. At the Mo cent re, estimated corrections calculated for the Fourier ripples artefact are v ery small when compared to the experimental associated errors, supporting t he suggestion that the fifth ligand is a water molecule rather than a hydro xide. Concerning the two iron-sulfur centres, asymmetry in the Fe-S distanc es as well as differences in the pattern of (NHS)-S-. . . hydrogen-bonding interactions was observed. which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each clu ster.