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1 Theoretical Study of Theoretical Study of Bioinorganic Reaction Bioinorganic Reaction Mechanisms Mechanisms Mireia Güell May 2007
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1
Theoretical Study of Theoretical Study of Bioinorganic Reaction Bioinorganic Reaction
MechanismsMechanisms
Mireia Güell
May 2007
2
• Bioinorganic Models of Non-heme Iron Oxygenases
• In collaboration with the Bioinorganic and Supramolecular Chemistry group, University of Girona
• Catalytic mechanism of Copper Enzymes
• In collaboration with the Theoretical Biochemistry group, University of Stockholm
_________________________________________________________________Introduction
Research topics
3
_________________________________________________________________Introduction
• Usually involved as redox catalysts in a wide range of biological processes
• Functions 1. metal ion uptake, transport and storage2. electron transfer3. dioxygen uptake, transport and storage4. catalysis
• Classification • Type-1 active site• Type-2 active site• Type-3 active site• Type-4 active site• The CuA active site
• The CuB active site
• The CuZ active site
Copper-containing proteins
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• Dicopper core
• Copper ions surrounded by 3 N atoms from 3 His residues
• Active site able to bind O2 at ambient conditions
• Cu(II) ions in the oxy state are strongly antiferromagnetically coupled
• Class represented by 3 proteins:
1. Hemocyanin2. Catechol oxidase3. Tyrosinase
_________________________________________________________________Introduction
Type-3 active site copper-containing proteins
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Catechol oxidase
_________________________________________________________________Introduction
• Type-3 active site copper enzyme
• Catalyses the oxidation of o-diphenols
• Found in plant tissues and crustaceans
• In 1998, Krebs and co-authors reported its crystal structure in 3 catalytic states:
• Native met (CuII-CuII) state• Reduced deoxy (CuI-CuI) state• Complex with inhibitor phenylthiourea
6
_________________________________________________________________Introduction
Catechol oxidase crystal structures
Native met (CuIICuII) stateComplex met (CuIICuII) state
with phenylthiourea
2.9 Å
Reduced deoxy (CuICuI) state
4.4 Å 4.2 Å
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Enzymatic reaction mechanism
_________________________________________________________________Introduction
8
_________________________________________________________________Introduction
Nat Struct Biol, 1998, 5,1084-1090
CuIAHis
His
His
BCuI His
His
His
H2O + H+
OH
OH
CuIIHis
His
His O
OCuII His
His
His
O
O
H+
OH
OH
O
O
H2O +
OH2
O2 +
O
HO
CuIIAHis
His
His
OH
BCuII His
His
His
O
HO
2H+
CuIIAHis
His
His
OH
BCuII His
His
His
met state
deoxy state
oxy state
A B+1
+2
+2
+3
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_________________________________________________________________Introduction
Cu
OH
Cu
His
His
His
His
His
His
CuHis
His
His O-
O-
Cu His
His
His
Cu
OH
Cu
His
His
His
His
His
HisO O
CuHis
His
His
Cu His
His
His
O2
2H+
OH
OH
CuHis
His
His O-
O-Cu His
His
His
O O
O
O
3H+2H+
OH
OH
O
O
H+
+ H2O
H2O +
Chem. Rev. 1996, 96, 2563-2605
0
+2
+1
+3
+2
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_________________________________________________________________Introduction
J Biol Inorg Chem, 2004, 9, 577-590
CuI CuI
His
His
His
His
His
His
O
O
H2O
H2O +
OHH
O
OH
O2
CuII CuI
His
His
His
His
His
HisO
O
OH
O
CuII CuI
His
His
His
His
His
HisO
O
O
OH
CuI CuI
His
His
His
His
His
HisO
O
OH
H
OH
CuI CuII
His
His
His
His
His
HisO
O
OH
H
OH
OH
OH
OH
OH
O
O
step astep e
step d step b
step c
+1+1
+1
+1 +1
CuIIHis
His
His O
OCuII His
His
His
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•Study the reaction mechanism of the following reaction:
• Objective
_________________________________________________________________Objective
CuIIHis
His
His O
OCuII His
His
His
with a model where:
• No protons leave or enter the active site during the catalytic cycle
• There is an intermediate with a µ−η2:η2 bridging peroxide
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_________________________________________________________________Methodology
•Model
• Methodology
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_________________________________________________________________Methodology
Cu Cu
His118
His88
His244
His274
His109Cys 92
His240
O
• Histidines Imidazoles
• Cysteine SCH3
• OH µ−η2:η2 bridging peroxide
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_________________________________________________________________Methodology
*
*
*
*
*
*
*
Total charge +2
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•Geometry optimizations
•B3LYP/lacvp
•For all stationary points Single point energy calculations B3LYP
•C,N,O,H,S cc-pvtz(-F)
•Cu lacv3p+
•Solvation effects
•B3LYP/lacpv
•solvation calculation using a Poisson-Boltzmann solver
•Outer dielectric constant of solvent = 4.0
•Radius of solvent probe molecule = 2.50 Å
_________________________________________________________________Methodology
• Computational details
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_________________________________________________________________
• Results
Results
1.681.71
17
-4.79
3
TS12
8.950.00
1
1.65
2
14.41
TS23
-4.43 TS34
-39.484
_________________________________________________________________Results
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_________________________________________________________________Results
OH
OH
O
O
+ O2 -2 + 2Cu2+ + 2OH- + 2Cu2+
The first half-reaction
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4.42
8
13.20TS67
2.78
TS56
_________________________________________________________________Results
0.006
2.08
5
-2.037
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_________________________________________________________________Results
The second half-reaction
OH
OH
O
O
+ 2OH - + 2Cu2+ + 2H2O + 2Cu+
21
7
6
2
_________________________________________________________________Results
1
5 4
3
Catechol Oxidase
Catalytic Cycle
22
_________________________________________________________________Conclusions
• Conclusions• A new mechanism for the catalytic cycle of the catechol oxidase
has been proposed, where:
• No external base is required since no protons leave or enter the active site during the catalytic cycle.
• There is an intermediate with a µ-η2:η2 bridging peroxide.
• The most critical step is the O-O cleavage, whose barrier (14.4 kcal/mol) is in reasonable agreement with the experimental rate (13 kcal/mol).
• In some steps there is a monodentate coordination of the substrate.
• The understanding of the catecholase activity of catechol oxidase can help to understand how tyrosinase works.
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_________________________________________________________________Conclusions
• Related projects...•Study of the catalytic mechanism of biomimetic complexes of catechol oxidase
Koval et al, J Biol Inorg Chem, 2005, 10, 739–750
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_________________________________________________________________Conclusions
Koval et al, Chem. Eur. J. 2006, 12, 6138 – 6150
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_________________________________________________________________Conclusions
•Study of the catalytic mechanism of of tirosinase
Matoba et al, J Biol Chem, 2006, 281, 8981–8990
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_________________________________________________________________Conclusions
•Study of the catalytic mechanism of biomimetic complexes of tirosinase
Palavicini et al, JACS 2005, 127, 18031-18036
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_________________________________________________________________Conclusions
Liviu M. Mirica, et al.Science, 2005, 308, 1890-1892
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•Prof Per Siegbahn and Prof. Margaretta Bloomberg…
… and all the members of the Theoretical Biochemistry group of the University of Stockholm
•All the members of the Institut de Química Computacional
•MEC
•The Swedish-Spanish Foundation for the Promotion of Education and Studies
_________________________________________________________________Acknowledgments
• Acknowledgments
