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(1)

Redox Chemistry of Hemoproteins

(Fe-Containing Enzymes)

Yoshitsugu Shiro

RIKEN SPring-8 Center

Japan

N

Fe

O

-N

N

Fe

O

(2)

To Understand Physiological Roles of Bio-metals Based on Structures of Metal-binding Proteins and Enzymes

Biometal Science Laboratory

城生体金属科学研究室

Redox Enzymes 酸化還元酵素 Sensor Proteins センサー蛋白質 Structural _Genomics 構造ゲノム科学

L

5

Fe

L

6 Axial Ligands

(3)

(xy, yz, xz) (x2_-y2_{, z}2₎

Octahedral

3d

Fe

2+

_(3d

6

₎

_Fe

₃₊

_(3d

₅

₎

High Spin S=2 Low Spin S=0 High Spin S=5/2 Low Spin S=1/2 none O H O CN

-Electronic Structure of Heme Iron

(4)

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.

L

5

Fe

L

6

(5)

Fe

L

6

N

H

δ+

_B

-δ−

His

Fe

3+

+ e

-

⇆ Fe

2+

Redox Potential

-300mV ～ +300mV

vs. NHE

(6)

Myoglobin (+50mV)

Fe

2+

L

6

N

H

O

His

(7)

Fe

2+

_Fe

2+

O

₂

O

₂

(8)

Fe

3+

N

H

+

O

His

O

-Asp

Peroxidases (-240mV)

2AH

₂

+ H

₂

O

₂

A

₂

H

₂

+ 2H

₂

O

(9)

Catalytic Mechanism of Peroxidases (Oxidation of Substrate AH₂) Fe3+ Fe4+ O H₂O₂ Fe4+ O AH2 .+ H₂O AH₂ AH. AH. Fe5+ O Compound I Peroxidases: Heme (Fe3+_): H₂O₂

[

O

]

+ H₂O (Heterolytic Cleavage) H₂O₂ HO･ + ･OH (Homolytic Cleavage)

(10)

Fe3+ N N -N N H N H NH₂ 2HN ₊ Fe3+ N N -N N H N H NH₂ 2HN ₊ O O H H

(11)

Fe3+ N N -N N H N H NH₂ 2HN ₊ O O H H + -Push Effect Pull Effect Fe5+ N N -N N H N H NH₂ 2HN ₊ O H₂O AH₂ e

(12)

-RH + O

₂

+ 2H

+

_{+ 2e}

-

_{ROH + H}

2

O

Cytochrome P450

Monooxygenase :

Fe

L

6

S

-Cys

Cytochrome P450

(13)

Fe

3+

H

₂

O

S

-Cys

Reductase

(-240mV)

e

-Low Spin (-300mV)

RH

High Spin (-170mV)

Fe

2+

S

-Cys

RH

(14)

Fe

2+

S

-Cys

RH

_O

2

Fe

3+

S

-Cys

RH

_O

2

･

Reductase

e

(15)

-Fe

3+

S

-Cys

RH

_O

2 -

H

2

O

H

+ solvent H-Bonding Network

Fe

3+

S

-Cys

RH

O

H

2

O

H

solvent H-Bonding Network

O

H

+

(16)

Fe

3+

S

-Cys

RH

O

H

O

H

Fe

5+

S

-Cys

RH

O

(17)

Fe

3+

S

-Cys

R

O

H

Fe3+ H₂O Fe3+ RH RH Fe2+ RH e -RH O Fe5+ O RH ROH H₂O₂ Shunt Pathway

(18)

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 Fe3+ H₂O NH₂ NH₂ HN + O -O C H₂O₂ H₂O Arg242

(19)

Fe3+ NH₂ NH₂ HN + O -O C H+ O -O H Fe3+ NH₂ NH₂ HN + O O C H O -HO H O

(20)

Fe5+ O NH₂ NH₂ HN + O -O C H H Fe3+ NH₂ NH₂ HN + O -O C H OH H₂O

(21)

Monooxygenase P450

Fungal NOR

(P450nor) Peroxygenase_P450

Overall Structure of P450 Type Enzymes

Reduction of Nitric Oxide：

2NO + 2H

+

_{+ 2e}

-

_{→ N}

(22)

Fe

3+

H

_NO

₂

O

Fe

3+

NO

CONH₂ H H N NADH H-_(H+ _{+ 2e}-₎

(23)

Fe

3+

NO

-

_H

+ CONH₂ H N NADH + Fe3+ N O e- _e -1851cm-1 530cm-1 Ferric 543cm-1 Fe2+ N O ? cm-1 Ferrous Fe3+ N O ~1330cm-1 596cm-1 Intermediate 2-反応中間体の電子構造（振動スペクトルから）

(24)

Fe

2+

NO

-

_H

+

ON

H

+

H

₂

O

solvent H-Bonding Network

Fe

3+

N

ON

HOH

(25)

Fe

3+

N

₂

O

H

₂

O

Fe3+ H₂O N₂O Fe3+ NO NO Fe2+ H +_{, 2e} -NO-_H+ N Fe3+ ONN OH ON H+ H₂O 脱窒カビ一酸化窒素還元酵素（P450nor）の反応機構

(26)

NO Reductase:

NO + (NO)

2-

_{+ 2H}

+

_{NNO + H}

2 O

Monooxygenase P450:

RH + (O

₂

)

2-

_{+ 2H}

+

_{ROH + H}

2 O

Peroxygenase P450:

RH + H

₂

O

₂

ROH + H

₂

O

反応中間体の電子構造

Fe

3+

NO

S

-

2-nH

+

(27)

Fe3+ NO

Cryo-Radiolysis (Reduction)

X-ray Fe3+ NO

2-?

?

Frozen Crystal

e

(28)

-Before X-ray exposure After X-ray exposure Fe3+ H₂O NO,H+ N₂O Fe3+ NO NO Fe2+ H +_{, 2e} -NO-_H+ Fe2+ N Fe3+ ONN OH ON H+ H₂O 脱窒カビ一酸化窒素還元酵素（P450nor）の反応機構

(29)

Denitrification

Nar:

Nir:

NOR:

Bacteria

Mo

Cu

or Fe

Heme(Fe)/Fe

Fungi

Mo

Cu

Heme(Fe)

チトクロムP450

Lateral Gene Transfer（水平遺伝子移動）

チトクロム酸化酵素

NOR Reaction

2NO + 2H

+

_{+ 2e}

-

_{→ N}

2

O + H

2

O

脱窒カビNOR:

水溶性

P450型

1 Heme(Fe)

脱窒菌NOR:

膜結合型

ｵｷｼﾀﾞｰｾﾞ型

3 Heme(Fe)+1 non-heme Fe

(30)

Heme c Fe His Met Heme bL His Fe His Heme bH Fe His Fenh His His His Cyt c e-複核活性中心

脱窒菌NOR

Binuclear Reaction Center

急速混合凍結装置

Liq.N₂ Mixer Frozen 100 μm Mixer (Expanded) Mixing-to-Freezing Time: 0.5 ~ 10 ms (Conventional:~ 100 ms） Reduced NOR NO

(31)

ESRスペクトル

Resting State 0.5 ms 1.0 ms 3.0 ms 10 ms M-to-F Time c bL bH Fe3+nh c bL bH Fe2+nh c bL bH Fe2+nh c bL bH Fe2+ nh c bL bH Fe3+ nh N N O O 4e-2NO 2H+ Intramolecular Electron Transfer 2NO N2O, H2O 2H+ 4e-O 休止状態完全還元型反応中間体１遷移状態部分酸化状態部分還元状態反応中間体１ ’ 完全酸化型 Fe3+ Fe3+ Fe2+ Fe2+ Fe2+ Fe3+ Fe2+ Fe2+ Fe2+ Fe3+ Fe3+ Fe2+ Fe2+ Fe3+ Fe3+ Fe3+ Fe3+ Fe3+ 提案した反応機構（作業仮説） Resting State

Fully Reduced State

Reaction Intermediate 1

Partially Reduced State Fully Oxidized State

(32)

Fe2+ _Fe2+ heme b₃ non-heme Fe_B N N O O 反応中間体（ミリ秒）

脱窒菌NOR

Fe3+ _Fe3+ heme b₃ non-heme Fe_B N N -_O O -? 繊維状態？（観測不能） 2H+

脱窒菌NOR

(33)

Fe3+ _Fe3+ heme b₃ non-heme Fe_B 酸化型 N₂O H₂O

脱窒菌NOR

2(NO

-

_{) + 2H}

+

_N

2 O + H

2 O

脱窒菌NORの反応

(34)

(ON)

2-

_NO

ON

-

_NO

--

_ON-NOH

H+

N

₂

O + H

₂

O

脱窒カビNOR 脱窒菌NOR

一酸化窒素還元（亜酸化窒素生成）の化学

Donor-Acceptor （供与ー受容型） Disproportionation （不均化型） Fe2+ _{N N} _Fe2+ O O Fe2+ _{N N} _Fe2+ O O

N-N 結合の生成／N-O 結合開裂

Reaction Intermediate Transient State

Fe-N-O の電子状態を精密に見たい振動分光（IR and Raman）法

(35)

酸素添加酵素

一原子酸素添加酵素

X + O

₂

+ 2H

+

_{+ 2e}

-

_{XO + H}

2

O

二原子酸素添加酵素

X + O

₂

XO

₂ N H X

O

₂ N H X O O H

ヘムを含む二原子酸素添加酵素

(36)

(37)

反応機構（作業仮説） Deprotonation By enzyme residue Deprotonation By oxygen Fe2+ O O N R H B Fe2+ O O N R H B Fe2+ O O N R H Fe2+ O O N R H Fe2+ O O N R H Fe2+ O O N R H O O +Trp