Speaker: Chen-Yang ZHOU
Supervisor: Yun-Dong WU

Google’s home page in 5.12: Dorothy Mary Hodgkin
Dorothy Mary Hodgkin, born in May 12, 1910
The 3 rd woman to win the Nobel Prize in
Chemistry (1964), for confirmation of the structure of penicillin and the Vitamin B
12 using X-ray crystallography

What is metalloprotein
Metal + Protein = Metallo protein
Protein containing a metal ion cofactor.
Estimated that ~ 1/2 of all proteins contain a metal.

• Example of 2 important reactions that matalloproteins play a central role
• The advantage of metals
• 1 detailed example: the importance of protein

Example 1: Photosynthesis
6CO
2
+ 12 H
2 light
O C
6
H
12
O
6
+ 6O
2
+6H
2
O
Oxygen evolution: 2H light
2
O 4e + 4H + + O
2
• Photosynthesis is the most important reaction in our planet
• CO
2 is converted into sugar, and generate O
2
• Photosynthesis fixes ~10 11 tons of carbon per year
• Oxygen evolution step is first key step, with metallo-oxo cluster as catalytic center
• Metallo-oxo cluster comprising 4 Mn n+ (n from 3 to 5) and 1 Ca
• Structure is controversial
• Cubane like, 2011, 1.9Å
Yasufumi Umena, Keisuke Kawakami, Jian-Ren Shen & Nobuo Kamiya. Nature 2011; 473: 55-60

Example 2: N
2
Fixation
• Nitrogen fixation: N
2 the atmosphere is converted into ammonia NH
• In industry : Haber-Bosch Process, high temperature and pressure
3
• In Nature: Nitrogenase
• 1.16Å structure resolved in 2002, but identification to the atom in the center is still uncertain. (until 2009)
Rhizobium （根瘤菌）
MoFeS -containing cofactor: breaking the N,N triple bond
Einsle O, Tezcan FA, Andrade SLA, Schmid B, Yoshida M, et al. 2002.
Science 297:1696 –700

What’s the advantage of metals
• Metallocofactors can bind gases
• Transition metals accept lone pair that act Lewis Acids
• Form coordination complex
Red blood cell
Myoglobin

What’s the advantage of metals
• Metallocofactors can act as super nucleophiles
S
N
2 reaction mechanism
Cob(I)alamin(vitamin B
12
):
supernucleophile

What’s the advantage of metals
• Radical-based reaction: biotin synthase
Dethiol biotin Biotin
• Radical-based reaction: halogenation by SyrB2
Blasiak, L. C., Vaillancourt, F. H., Walsh, C. T. and Drennan, C. L. Nature 2006, 440, 368–371

Detailed Example: Acetogenesis
• Carbon Fixation Without Oxygen
• Acetogenesis fixes ~10 10 tons of carbon per year,
10% of total
• One of the six known pathways for carbon fixation
(Wood-Ljungdahl pathway)
• Energy efficient: lowst net ATP requirement
• O
2
-sensitive: Fe-S, Ni-Fe-S clusters (anaerobic)

Acetogenesis: Wood-Ljungdahl pathway
CH
3
-
H
4 folate
CO
CO
2

Metallo-centers in Acetogenesis
• Key to acetogenesis are protein-bound NiFeS -containing and Co -containing cofactors
Binds and transfers
CH
3
Binds and reduces
CO
2 to CO
Assembles the acetate
S.W. Ragsdale, E. Pierce, Biochimica et Biophysica Acta 2008; 1784: 1873–1898 (Review)

Zoom in to the Methyl Transfer Step
Vitamine B
12 derivative,
Cobalamin, is a supernucleophile
They form a complex
Methyl-transferase
Corrinoid FeS
Protein

Protein Structures in Methyl Transfer Step
83kDa
56kDa
Why such an elaborate protein
framework ( 140kDa ) is required for such a simple, yet biologically essential reaction?
Ando N, Kung Y, Can M, Bender G, Ragsdale SW, Drennan CL. (2012) J. Am. Chem. Soc. 134 (43) 17945 –17954.

Protein Structures in Methyl Transfer Step
The distance for the reactant is ~25Å
Swinging move ~7Å towards the CH
3
1. B
12 must be uncapped to do
S
N
2 substitution, and move a distance of ~18Å
2. Is the crystal structure active?

Is the Crystal active?
Different valance state of
Co has different color control group
Result of ultraviolet– visible absorption spectroscopy
Kung Y, et al. 2012. Nature 484:265–269.

Folate induce clamping motion: ~17Å
Resolution of crystal structures
Folate-free: 2.38 Å
Folate-bound: 3.50 Å
Kung Y, et al. 2012. Nature 484:265–269.

The last move of B
12
2Fo-Fc map in blue mesh (1.0 σ)
Fo-Fc difference map in green mesh (3.0 σ)
Anomalous difference map in pink mesh (4.0 σ)
Intriguingly, a large, continuous electron density peak is present in 2 F o
− F c
, F o
− F c
, and composite omit maps, emanating from the corrin ring and stretching directly over the folate-binding site,

Cartoon model of B
12
CFeSP/MeTr
-dependent methyl transfer in
Kung Y, et al. 2012. Nature 484:265–269.

• Protein protect the reactive B
12 before and after CH
3 transfer by capping domain
• The conformational change is controlled by protein, which send the B
12 to the CH
3
-H
4 folate.
• The crystal complex is active, and the conformational change can happen even in the crystal lattice. (The largest conformational change observed in crystal.)
• Protein scaffold and metallo center depend on each other to have amazing reactivity and selectivity