RESEARCH ACTIVITIES II Department of Molecular Structure II-D Structure and Function of Metalloproteins and Its
Molecular Design
Metal ion is a common cofactor that is crucial for active centers of proteins involved in many biologically important processes in cells, and a relatively small number of metal-based prosthetic groups are utilized to servenumerous and diverse chemical functions. A typical metal-based prosthetic group, which represents a fascinatingexample in this respect, is heme. Heme promotes a variety of functions, such as dioxygen storage, activation ofsmall molecules, electron transfer reactions, and sensing gaseous molecule. In the field of protein design andengineering, hemoproteins also make particularly attractive targets. There are many reasons for this, including theexciting possibility of engineering protein-based molecules with useful catalytic, electronic or optoelectronicproperties. Based on various kinds of spectroscopies, we have functionally and structurally characterized somehemoproteins including newly identified heme-regulated proteins, and designed hemoproteins showing improvedactivities and new functions.
II-D-1 L358P Mutation on P450cam Simulates
II-D-2 Structural Diversities of Active Site in
Structural Changes upon Putidaredoxin
Clinical Azole Bound Forms between Sterol
Binding. The Structural Changes Trigger
14α-demethylases (CYP51) from Human and
Electron Transfer to Oxy-P450cam from
Mycobacterium tuberculosis
Electron Donors
TOSHA, Takehiko1; YOSHIOKA, Shiro1;
Takehiko; HORI, Hiroshi2; ISHIMORI, Koichiro3;
(1Kyoto Univ.; 2IMS and Kyoto Univ.) KAGAWA, Norio4; WATERMAN, Michael R.4
(1Kyoto Univ.; 2Osaka Univ.; 3IMS, Kyoto Univ. and
[J. Biol. Chem. 279, 42836–42843 (2004)]
Hokkaodo Univ.; 4Vanderbilt Univ.) To investigate the functional and structural charac- [J. Biol. Chem. 280, 9088–9096 (2005)]
terization of a crucial cytochrome P450cam (P450cam)-putidaredoxin (Pdx) complex, we utilized a mutant To gain insights into the molecular basis of the whose spectroscopic property corresponds to the proper- design for the selective azole anti-fungals, we compared ties of the wild type P450cam in the presence of Pdx.
the binding properties of azole-based inhibitors for The 1H NMR spectrum of the carbonmonoxy adduct of cytochrome P450 sterol 14α-demethylase (CYP51) the mutant, the Leu-358 3 Pro mutant (L358P), in the from human (HuCYP51) and Mycobacterium tuberculo- absence of Pdx showed that the ring current-shifted sis (MtCYP51). Spectroscopic titration of azoles to the signals arising from D-camphor were upfield-shifted CYP51s revealed that HuCYP51 has higher affinity for and observed as resolved signals, which are typical for ketoconazole (KET), an azole derivative that has long the wild type enzyme in the presence of Pdx. Signals lipophilic groups, than MtCYP51, but the affinity for from the β-proton of the axial cysteine and the γ-methyl fluconazole (FLU), which is a member of the anti- group of Thr-252 were also shifted upfield and down- fungal armamentarium, was lower in HuCYP51. The field, respectively, in the L358P mutant as observed for affinity for 4-phenylimidazole (4-PhIm) to MtCYP51 Pdx-bound wild type P450cam. The close similarity in was quite low compared with that to HuCYP51. In the the NMR spectra suggests that the heme environment of resonance Raman spectra for HuCYP51, the FLU bind- the L358P mutant mimics that of the Pdx-bound en- ing induced only minor spectral changes, whereas the zyme. The functional analysis of the L358P mutant has prominent high frequency shift of the bending mode of revealed that the oxygen adduct of the L358P mutant the heme vinyl group was detected in the KET- or 4- can promote the oxygenation reaction for D-camphor PhIm-bound forms. On the other hand, the bending with nonphysiological electron donors such as dithionite mode of the heme propionate group for the FLU-bound and ascorbic acid, showing that oxygenated L358P is form of MtCYP51 was shifted to high frequency as “activated” to receive electron from the donor. Based on found for the KET-bound form, but that for 4-PhIm was the structural and functional characterization of the shifted to low frequency. The EPR spectra for 4-PhIm- L358P mutant, we conclude that the Pdx-induced struc- bound MtCYP51 and FLUbound HuCYP51 gave multi- tural changes in P450cam would facilitate the electron ple g values, showing heterogeneous binding of the transfer from the electron donor, and the Pdx binding to azoles, whereas the single gx and gz values were ob- P450cam would be a trigger for the electron transfer to served for other azole-bound forms. Together with the alignment of the amino acid sequence, these spectro-scopic differences suggest that the region between theB’ and C helices, particularly the hydrophobicity of theC helix, in CYP51s plays primary roles in determiningstrength of interactions with azoles; this differentiatesthe binding specificity of azoles to CYP51s.
II-D-3 Two Heme Binding Sites Are Involved in
in its recognition by the HOIL-1 ubiquitin ligase. Al- the Regulated Degradation of the Bacterial Iron
though HRMs are known to sense heme concentration Response Regulator (Irr) Protein
by simply binding to heme, the HRM in IRP2 specifical-ly contributes to its oxidative modification, its recogni- YANG, Jianhua1; ISHIMORI, Koichiro2; O’BRIAN,
tion by the ligase, and its sensing of iron concentration (1New York State Univ.; 2IMS and Kyoto Univ.) [J. Biol. Chem. 280, 7671–7676 (2005)]
The iron response regulator (Irr) protein from Bradyrhizobium japonicum is a conditionally stableprotein that degrades in response to cellular iron avail-ability. This turnover is heme-dependent, and rapiddegradation involves heme binding to a heme regulatorymotif (HRM) of Irr. Here, we show that Irr confers iron-dependent instability on glutathione S-transferase (GST)when fused to it. Analysis of Irr-GST derivatives withC-terminal truncations of Irr implicated a second regionnecessary for degradation, other than the HRM, andshowed that the HRM was not sufficient to confer insta-bility on GST. The HRM-defective mutant IrrC29Adegraded in the presence of iron but much more slowlythan the wild-type protein. This slow turnover washeme-dependent, as discerned by the stability of Irr in aheme-defective mutant strain. Whereas the HRM ofpurified recombinant Irr binds ferric (oxidized) heme, asecond site that binds ferrous (reduced) heme wasidentified based on spectral analysis of truncation andsubstitution mutants. A mutant in which histidines117–119 were changed to alanines severely diminishedferrous, but not ferric, heme binding. Introduction ofthese substitutions in an Irr-GST fusion stabilized theprotein in vivo in the presence of iron. We conclude thatnormal iron-dependent Irr degradation involves twoheme binding sites and that both redox states of hemeare required for rapid turnover.
II-D-4 Involvement of Heme Regulatory Motif in
Heme-Mediated Ubiquitination and Degradation
of IRP2

ISHIKAWA, Haruto1; KATO, Michihiko2; HORI,
Hiroshi3; ISHIMORI, Koichiro4; KIRISAKO,
Takayoshi2; TOKUNAGA, Fuminori2; IWAI,
(1Osaka City Univ.; 2Osaka City Univ. and CREST;
3Osaka Univ.; 4IMS, Kyoto Univ., CREST and
Hokkaodo Univ.)

[Mol. Cell. 19, 171–181 (2005)]
Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degra-dation. We have shown that IRP2 degradation is trig-gered by heme-mediated oxidation. We report here thatnot only Cys201, an invariant residue in the heme regu-latory motif (HRM), but also His204 is critical for IRP2degradation. Spectroscopic studies revealed that Cys201binds ferric heme, whereas His204 is a ferrous hemebinding site, indicating the involvement of these resi-dues in sensing the redox state of the heme iron and ingenerating the oxidative modification. Moreover, theHRM in IRP2 has been suggested to play a critical role


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