Redox Chemistry of emoproteins (Fe-Containing Enzymes) Yoshitsugu Shiro RIKE SPring-8 Center Japan eme (Fe-Porphyrin Complex) Ō Fe - Provides 4 itrogen Atoms as Planar Ligands 1
Biometal Science Laboratory 城生体金属科学研究室 Sensor Proteins センサー蛋白質 Redox Enzymes 酸化還元酵素 Structural Genomics 構造ゲノム科学 To Understand Physiological Roles of Bio-metals Based on Structures of Metal-binding Proteins and Enzymes L6 Fe L5 Axial Ligands 2
(x 2 -y 2, z 2 ) 3d (xy, yz, xz) ctahedral Electronic Structure of eme Iron (3d 6 ) (3d 5 ) igh Spin S=2 Low Spin S=0 igh Spin S=5/2 Low Spin S=1/2 L6 none 2 C 2 C - F - - 3-3
In Metal-Binding Proteins, 1. The Properties of the Metal Ions Can Be Modulated by the Protein Part. 2. The Protein Part Can Support the Metal Reactivity. L6 Fe L5 Axial Ligands 4
L6 Fe is δ δ+ B - Redox Potential + e - -300mV ~ +300mV vs. E 5
Myoglobin (+50mV) L6 is Myoglobin (+50mV) 6
Reversible xygen Binding in Ferrous State 2 2 Peroxidases (-240mV) 7
- is + - Peroxidases (-240mV) Asp xidation of Substrate (A 2 ) by 2 2 2A 2 + 2 2 A 2 2 + 2 2 8
Catalytic Mechanism of Peroxidases (xidation of Substrate A 2 ) A. 2 2 Fe 4+ A 2 A 2 2.+ Fe 4+ Compound I A. Fe 5+ - Bond Cleavage Peroxidases: 2 2 [] + 2 (eterolytic Cleavage) eme ( ): 2 2 + (omolytic Cleavage) 9
2 + 2 - - 2 + 2 10
+ Pull Effect 2 + - 2 - Push Effect e - A 2 2 + 2 2 Fe 5+ - 11
Cytochrome P450 Monooxygenase : R + 2 + 2 + + 2e - R + 2 L6 Fe S - Cys Cytochrome P450 12
R 2 Cys S - Low igh Spin (-300mV) (-170mV) e - Reductase (-240mV) R Cys S - 13
R 2 Cys S - R 2 Cys S - e - Reductase 14
R 2 - + 2 -Bonding etwork solvent S - Cys R + 2 -Bonding etwork solvent S - Cys 15
R Cys S - R Fe 5+ Cys S - 16
R Cys S - Reaction Mechanism of Cytochrome P450 2 R R R 2 2 e - R Shunt Pathway R Fe 5+ R 2 2 2 +, e - 17
一原子酸素添加酵素チトクロム P450 脂肪酸水酸化酵素ペルオキシゲナーゼ P450 C - Arg242 + 2 2 2 2 2 2 18
C - + 2 2 - + C - + 2 2 2 19
C - + 2 2 Fe 5+ C 2 - + 2 2 20
verall Structure of P450 Type Enzymes Monooxygenase P450 Fungal R (P450nor) Peroxygenase P450 Reduction of itric xide: 2 + 2 + + 2e - 2 + 2 21
2 - ( + + 2e - ) AD C 2 22
+ AD - + C 2 反応中間体の電子構造 ( 振動スペクトルから ) 1851cm -1 530cm -1? cm -1 543cm -1 2- ~1330cm -1 596cm -1 Ferric e - e - Ferrous Intermediate 23
solvent -Bonding etwork 2 + - + 24
2 2 脱窒カビ一酸化窒素還元酵素 (P450nor) の反応機構 2 2 +, 2e - 2 - + +, + 25
Monooxygenase P450: R + ( 2 ) 2- + 2 + Reductase: + () 2- + 2 + Peroxygenase P450: R + 2 2 R + 2 R + 2 + 2 反応中間体の電子構造 2- n + S - 26
Cryo-Radiolysis (Reduction) Frozen Crystal e - X-ray????? 2-?? 27
Before X-ray exposure After X-ray exposure 脱窒カビ一酸化窒素還元酵素 (P450nor) の反応機構 2 2 +, 2e - 2 - + +, + 28
ar: ir: Denitrification Bacteria Mo Cu or Fe Fungi Mo Lateral Gene Transfer( 水平遺伝子移動 ) Cu R: eme(fe)/fe eme(fe) チトクロム酸化酵素 チトクロム P450 R Reaction 2 + 2 + + 2e - 2 + 2 脱窒カビ R: 水溶性 P450 型 1 eme(fe) 脱窒菌 R: 膜結合型オキシタ ーセ 型 3 eme(fe)+1 non-heme Fe 29
脱窒菌 R is Fe Met e - Cyt c eme c e - is Fe is eme b L e - is Binuclear Reaction 複核活性中心 Center Fe eme b Fe nh is is is 急速混合凍結装置 Mixer (Expanded) 100 μm Mixer Reduced R Liq. 2 Frozen Sample Mixing-to-Freezing Time: 0.5 ~ 10 ms (Conventional:~ 100 ms) Designed by Dr. S. Takahashi (saka Univ.) 30
ESR スペクトル Resting State M-to-F Time 0.5 ms 1.0 ms 3.0 ms 10 ms 休止状態 Resting State c Fe3+ Fe3+ nh b L b Fully xidized State 完全酸化型反応中間体 1 2 + c c b L b b L b Fe3+ nh Fe2+ nh 4e - 4e - 2, 2 2 c 完全還元型 Fully Reduced State Fe2+ Fe2+ nh b L b 提案した反応機構 ( 作業仮説 ) 部分還元状態 Partially Reduced State c Fe2+ Fe2+ nh b L b 2 Intramolecular Electron Transfer Reaction Intermediate 1 反応中間体 1 遷移状態部分酸化状態 c c c Fe2+ Fe2+ nh Fe3+ Fe3+ nh Fe3+ Fe3+ nh b L b b L b b L b 2 + 2, 2 31
脱窒菌 R heme b 3 non-heme Fe B 反応中間体 ( ミリ秒 ) 脱窒菌 R 2 + - - heme b 3 non-heme Fe B? 繊維状態? ( 観測不能 ) 32
脱窒菌 R 2 2 heme b 3 non-heme Fe B 酸化型 脱窒菌 R の反応 2( - ) + 2 + 2 + 2 33
一酸化窒素還元 ( 亜酸化窒素生成 ) の化学 脱窒カビ R () 2- Donor-Acceptor ( 供与ー受容型 ) - - + 2 + 2 脱窒菌 R - - Disproportionation ( 不均化型 ) - 結合の生成 /- 結合開裂 Reaction Intermediate Transient State Fe-- の電子状態を精密に見たい振動分光 (IR and Raman) 法 34
酸素添加酵素一原子酸素添加酵素 X + 2 + 2 + + 2e - X + 2 二原子酸素添加酵素 X + 2 X 2 ヘムを含む二原子酸素添加酵素 X 2 X 35
インドールアミン 2,3 ジオキシゲナーゼ 36
反応機構 ( 作業仮説 ) Deprotonation By enzyme residue B R B R R Deprotonation By oxygen +Trp R R R In Metal-Binding Proteins, 1. The Properties of the Metal Ions Can Be Modulated by the Protein Part. 2. The Protein Part Can Support the Metal Reactivity. 37
蛋白質構造解析の分解能は? 構造変化を理解する 低 触媒反応を理解する 電子状態を理解する 高 38