Structural biology relating to electron transfer

Most electron transfer proteins have cofactor molecules, such as heme, flavin, and iron-sulfur clusters. Electron transfer reactions are regulated by the redox states of the cofactors, and hydrogen atoms are often involved in the conversion of redox states. Therefore, it is important to obtain precise structure information including hydrogen atoms for understanding the electron transfer reactions. We have determined structures of some electron transfer proteins, cytochrome b5 (b5; binding a heme cofactor) and NADH-cytochrome b5 reductase (b5R; binding a FAD cofactor) at the resolutions higher than 1.0 Å by X-ray crystallography. Structure information of some hydrogen atoms were clarified by these X-ray analyses. On the other hand, the rest of the hydrogen positions are still ambiguous.Neutron crystallography is a powerful technique to obtain accurate positions of hydrogen atoms in protein structures. Recently, we have performed high-resolution neutron and X-ray crystal structure analyses of b5R. b5R catalyzes the electron transfer from two electron carriers of NADH to one electron carrier of b5 and participates in fatty acid synthesis, cholesterol synthesis, and xenobiotic oxidation as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, b5R also participates in the reduction of methemoglobin. We succeeded in data collection of b5R (oxidized form) at high resolutions, 1.40 Å (at iBIX in J-PARC) and 1.45 Å (at BIODIFF in FRM-II), under cryogenic conditions. We have observed a hydrogen bonding network from FAD to His49, which is the only polar residue in a cluster of hydrophobic residues on the surface near FAD, so it seems to be responsible for electron transfer to b5. In addition, we have determined high-resolution X-ray crystal structures of the reduced form of b5R using wild-type and T66V mutant. The electron density map of the NAD cofactor clearly displays NAD+ and NADH states in wild-type and mutant, respectively. The neutron and X-ray structure analyses provide information about the hydrogen transfer pathway in b5R.3rd QST International Symposiu