Projecte tesi

Theoretical Study of Bioinorganic Reaction Mechanisms Mireia Güell May 2007

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

_________________________________________________________________ Introduction
Usually involved as redox catalysts in a wide range of biological processes
Functions
metal ion uptake, transport and storage
electron transfer
dioxygen uptake, transpor t and storage
catalysis
Classification
Type-1 active site
Type-2 active site
T ype-3 active site
Type-4 active site
The Cu A active site
T he Cu B active site
The Cu Z active site
Copper-containing proteins

Dico p per core
Co p per ions surrounded by 3 N atoms from 3 His residues
Active site able to bind O 2 at ambient conditions
Cu(II) ions in the oxy state are strongly antiferromagnetically coupled
Class represented by 3 proteins:
Hemocyanin
Catechol oxidase
Tyrosinase
_________________________________________________________________ Introduction Type-3 active site copper-containing proteins

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 (Cu II -Cu II ) state
Reduced deoxy (Cu I -Cu I ) state
Complex with inhibitor phenylthiourea

_________________________________________________________________ Introduction Catechol oxidase crystal structures Native met (Cu II Cu II ) state Complex met (Cu II Cu II ) state with phenylthiourea Reduced deoxy (Cu I Cu I ) state 4.2 Å 2.9 Å 4.4 Å

Enzymatic reaction mechanism _________________________________________________________________ Introduction

_________________________________________________________________ Introduction Nat Struct Biol, 1998, 5,1084-1090 +1 +2 +2 +3

_________________________________________________________________ Introduction Chem. Rev. 1996, 96, 2563-2605 0 +2 +1 +3 +2

_________________________________________________________________ Introduction J Biol Inorg Chem, 2004, 9, 577-590 +1 +1 +1 +1 +1

Study the reaction mechanism of the following reaction:
Objective
_________________________________________________________________ Objective with a model where:
No protons leave or enter the active site during the catalytic cycle
There is an intermediate with
a        bridging peroxide

_________________________________________________________________ Methodology
Model
Methodology

_________________________________________________________________ Methodology Cu Cu His118 His88 His244 His274 His109 Cys 92 His240 O
Histidines  Imidazoles
Cysteine  SCH 3
OH         bridging peroxide

_________________________________________________________________ Methodology * * * * * * * Total charge  +2

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

_________________________________________________________________
Results
Results 1.68 1.71

0.00 1 _________________________________________________________________ Results -4.79 3 TS12 8.95 1.65 2 14.41 TS23 -4.43 TS34 -39.48 4

_________________________________________________________________ Results
The first half-reaction

_________________________________________________________________ Results 0.00 5 4.42 8 13.20 TS67 2.78 TS56 6 2.08 -2.03 7

_________________________________________________________________ Results
The second half-reaction

_________________________________________________________________ Results 1 Catechol Oxidase Catalytic Cycle 7 6 2 5 4 3

_________________________________________________________________ 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  -  2 :  2 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.

_________________________________________________________________ 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

_________________________________________________________________ Conclusions Koval et al, Chem. Eur. J. 2006, 12, 6138 – 6150

_________________________________________________________________ Conclusions
Study of the catalytic mechanism of of tirosinase
Matoba et al, J Biol Chem, 2006, 281, 8981–8990

_________________________________________________________________ Conclusions
Study of the catalytic mechanism of biomimetic complexes of tirosinase
Palavicini et al, JACS 2005, 127, 18031-18036

_________________________________________________________________ Conclusions Liviu M. Mirica, et al. Science, 2005, 308, 1890-1892

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

Projecte tesi

by mguellse on Nov 06, 2009

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