Iron is an absolute requirement for nearly a]1 organ{sms. Bacterial pathogens haveevolvedtheironacquisitionsystemsbecauseironisnotreadilyavailablein human body, Staphyloeoccou.g aurezas emp]oys the iron-Teguiated surface determinant (Isd) systern as its primary heme-iron uptake pathway. In other words, this system is a promising target to battle S.aureus. Heme molecule is first extracted frorn humans' hemoglobin and captured by IsdH or IsdB. IsdA and IsdC relays heme to IsdE on the plasrna

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... ane. Although the crystal stmctures of Isd proteins have been so]ved with and without heme, the mechanism of the one-way transport is still unclear because of the close similarity of these structures. We examined the herne-binding and hemetransport mechanisms of Isd proteins not on]y in vitro, but also using in silico methodologies. Isothermal titration calorimetry {ITC) experiments showed that the downstream Isd proteins have higher acanities against heme than the upstream proteins. Qualitatively consistent results were obtained from the freeenergy calculations with the MM-PBSA method, which revealed enthalpic contributjons ofthe residues te the asunity to heme for each Isd protein. Based on these results, we designed and synthesized a beme derivative. We wM examine its growth inhibition activity in vivo. In addition, the QMIMM (ONIOM} caleulation is being perfOrmed for a model of a heme-transport complex composed of two Isd proteins and one heme. We wi[] discuss the transport rnechanism between the Isd proteins. t F.NEyNeyopMJmaelmtsmNml[aslt6tsfi : vamasm. =-n mmEttl[nmuTree4Fe-Hisreaopett Essence in oxygen affinity regulation of human adlllt hemogtobin: Change of Fe-His bond tength by quaternary or tertiary structure change Nagatome, Yasuhisa Yamamura, Kazuya Saito (Dept. Chem., Uhiv. shows some IHP-induced hyper fine shifted (hfs) signals besides the heme methyl signals around 15-35 ppm. The addition of L3S to the N3-metHb with IHP showed that the heme signals were shifted and broadened, but the IHPinduced hfs signals were increased. The pH dependence ofthe hfs signals was also examined and the pH increase from 6 to 8 showed that the IHP-induced signals disappeared, suggesting that the spectral change should be related to the change in the oxygen affinity. The quaternary structural change ofmetHb will bc discussed based on hydrogen bond signals areund 12-13 ppm. 2PT154 Momoko Ichii2, Centerfi)rM Llosei Uhiv.) e F.NEaU ifYOp 145Tyr ut Thr lte(; Leu ,NO-Mffzama emmt[sx6rel Effects of the substitlltions of Thr or Leu for fi145 Tyr on the structure and oxygen binding properties of human hemoglobin Yukifumi Nagaii, Kiyohiro Imaib'2, Masako Nagaii (tRes. icro-Ndno Tlich.Hbsei Univ., !bontier Biosci. andARpiied Chem. 2PT152 ShigenoriZrukuba)Hurnan adult hemoglobin (HbA) possessing a2 P2 tetramer structure exhibits coeperativity in oxygen bincling. X-ray crystallegraphie studies have demonstrated the presence of two distinct quaternary structures, cal]ed T (tense) and R (rclaxed) states, which correspond to the ]owand high-02 arnnity forms, respectively, Both the quaternary structures ofT and R exhibit different Fe-His bond lengths/ they are shorter in R state than T state in both the a and P subunits. Extensive examinations of HbA in the presence of the atlosteric effeetor bezafibrate (BZF) showed a reduction ofthe oxygen asunity ofoxyHbA in the R structure and the importance oftertiary structure change foT oxygen affinity is stressed. Recently, one ofauthors reported that the decrease in oxygen affinity ofoKyHbA upon BZF addition accompanies the elongation of Fe-His bond in a subunit[1],Ourfindingisconsistentwiththemoleculardynamicscalculationof Yonetani and Laberge [2], in which the main chain in E and F helices of HbA fluctuates more in the pTesence than in the absence of BZF. This enhanced fluctuation in the E and F helices results in weakening of the Fe-His bond. Thus, the longation of Fe-His bond length is caused by not only quaternary structure but also tertiary structure change. This suggests that the e]ongation of Fe-His bond is essential in oxygen affinity regulation of human adu]t hemoglobin. [1] S. Nagatomo, H, Hamada and H. Yoshikawa, J. Phys. Chem. B 11S, 12971 (20i1). [2] T. Yonetani and M. Laberge, Biochim. Bioph.ys. Acta 1784, 1146 {2008). 2PT153 Structural fluct"ation and change around heme pockets of liganded Hb induced by L35 binding Kenji Kanaorii, Yusuke Tajirii, Antonio Tsuneshige2, Takashi Yonetani3 (iGrad. Sch., 1<yoto Jnst. of'fech., !Hosei Uhiv" iUniv. Pennsyivania) Hemoglobin (Hb) takes two struetures, depending on oxygen binding; Tquaternary structure with low oxygen-arnnity and R-quaternary structure with high exygen-acanity. Heterotropic effectors deerease the oxygen-affinity and its cooperativity. In the presence of two potent effectors, L35 and inositot hexaphosphate (IHP) at pH 6.8. 1H-NMR spectra of human ad"lt Hb showed oKy-T-quatennary structure. The NMR signals around heme pockets of the oxy-T structure were rather broad, indicating that some structura] fiuctuations occur around the herne, and the fluctuations may be related with the reduction of the oxygen-aennity, In order to clarify the structural fiuctuations in the hemepocket induced by L35 and IHP, NMR spectra of tiganded met-Hbs were measured in the presence ofthe effectors. It has been known that the N3-metHb In all the vertebrate hemog]obins and rnyoglobins, the penultimate position (HC2) is invariably occupied by tyrosine (Tyr}. The natural and anificial mutants of P14STyr studied so far, show the extremely high oxygen athnity and low cooperativity, To examine the role of B 145Tyr on the structure and oxygen binding properties, we expressed two new artificial mutant Hbs {n E. coli, rHb(6Y145L) and rHb{PY145T) and purified thern as described previous]y [1]. These mutant Hbs exhibited a markedly high exygen athnity and dirninished cooperativity (Pse=O.6-O,9 mmHg, Hi]1's n=1.3-1.4). In the presence of inisitol hexaphesphate (IHP), however, oxygen affinity of these mutarit Hbs decreased and exhibited significant cooperativity; Pso=1,7 mrnHg, n=: 1.9 for Hb {PY145T); Pso=2.4 mmHg, n=2,2 for Hb(PY]45L). From the oxygen binding properties of these rnutant Hbs, the replacement of P14STyr by Leu seerns to stabilize the T-conformation more than that of Thr, DeoxyHb(BYt4SL) exhibited a smalter negative CD band at 286 nm (a T-state rnarker band) than that of deoxyHbA, but exhibited an increase ln intensity of the negative CD band upen the addition of IHP, These results indicate that the residue at HC2 of P subunits contributes to stabi]ization of the T-conformation through hydrophobic interactions with the surrounclings in addition to hydrogen bonding to