Charge-density analysis of an iron–sulfur protein at an ultra-high resolution of 0.48 Å

Abstract

The fine structures of proteins, such as the positions of hydrogen atoms, distributions of valence electrons and orientations of bound waters, are critical factors for determining the dynamic and chemical properties of proteins. Such information cannot be obtained by conventional protein X-ray analyses at 3.0-1.5 Å resolution, in which amino acids are fitted into atomically unresolved electron-density maps and refinement calculations are performed under strong restraints. Therefore, we usually supplement the information on hydrogen atoms and valence electrons in proteins with pre-existing common knowledge obtained by chemistry in small molecules. However, even now, computational calculation of such information with quantum chemistry also tends to be difficult, especially for polynuclear metalloproteins. Here we report a charge-density analysis of the high-potential iron-sulfur protein from the thermophilic purple bacterium Thermochromatium tepidum using X-ray data at an ultra-high resolution of 0.48 Å. Residual electron densities in the conventional refinement are assigned as valence electrons in the multipolar refinement. Iron 3d and sulfur 3p electron densities of the Fe4S4 cluster are visualized around the atoms. Such information provides the most detailed view of the valence electrons of the metal complex in the protein. The asymmetry of the iron-sulfur cluster and the protein environment suggests the structural basis of charge storing on electron transfer. Our charge-density analysis reveals many fine features around the metal complex for the first time, and will enable further theoretical and experimental studies of metalloproteins.

Crystallographic characterization of the high-potential iron-sulfur protein in the oxidized state at 0.8 Å resolution

“…Our results for the difference in the Fe-S lengths implies that the subcluster composed of Fe1, Fe2, S3, and S4 is important for storing electronic charges in HiPIP. This implication is consistent with the previous results such as charge-density analysis [18] and X-ray absorption spectroscopic analysis [32]. In addition, most distances between the sulfur atoms of the iron-sulfur cluster and the protein environment are elongated upon the oxidation (Table 4).…”

“…In addition, the bond lengths of Fe- (Cys-S γ ) for all Fe atoms are also decreased by the oxidation. The almost identical redox-related structural changes are observed for the comparison to the reduced structure at 0.48 Å, which were refined with the multipolar atomic model [18]. The previous X-ray crystallographic analysis of HiPIP from A .…”

“…We have performed high-resolution crystallographic investigations of HiPIP from a thermophilic purple bacterium Thermochromatium tepidum [14–17]. In addition, we recently succeeded in performing a charge-density analysis of HiPIP in the reduced state at 0.48 Å resolution [18]. In order to reveal detailed structural differences between the reduced and oxidized states, a high-resolution determination of the structure in the oxidized HiPIP is indispensable.…”

Mapping elusive electron density

“…Even the simplest iron-sulfur electron transfer proteins are structurally and electronically complex 2 , and the electron transfer (redox) potential is tuned by the protein and solvent surrounding the iron-sulfur active site [3][4][5] . A study appearing in Nature that reports an ultra-high-resolution structure of an iron-sulfur protein involved in bacterial photosynthesis now offers these insights 6 . This network is particularly important for modulating the redox potential of the protein, and it acts as an electron density accepting region from the cluster anion 4,5 .…”

Ultra–high‐resolution structure and charge‐density analysis of high‐potential iron–sulfur protein

“…Accordingly, we have continued further crystallographic analyses of HiPIP to perform charge-density analyses that have never been applied to any metalloproteins. Finally, we successfully determined its crystal structure at an ultrahigh (0.48 A) resolution, which enabled us to perform a charge-density study to visualize the distribution of valence electrons in this metalloprotein [14].…”