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IntroductionOur work is aimed at making hybrid myoglobins to use in photochemical studies of catalysis by heme proteins. Although myoglobin does not perform enzymatic functions in vivo, catalytically active analogues can be synthesized via the modification of the heme group (porphyrin) with a photoactive ruthenium bpy pendant arm. Reconstitution of these into apomyoglobin serves as a model for the catalytically active heme proteins.
Mb active site
Myoglobin
Synthesis of Bpy Pendant Arm
LDA, Br(CH) 6Br
THF DMF
KN
O
O
N N N N
(CH2)7Br
N N
(CH2)7 N
O
O
Conc. HClReflux, 10 hours
N N
(CH2)7NH2
N N
(CH2)NH250% NaOH/sec-BuOH
Free base
85% 87%
84%
•Using this method, we cansystematically vary the length and composition of the pendant arm.
Synthesis of Protoporphyrin IX Derivative
NNH
N HN
CO2Et CO2H
THFPCl5, EtOHNNH
N HN
CO2Na CO2Na
sep. on SiO2
35%
•This protects one proprionate which will be nessasary to help stabilize our reconstituted protein.
The AmideCoupling Reaction
NNH
N HN
CO2Et CO2H
N N
(CH2)NH2
+
DECP, (Et) 3NTHF
Reflux 48 hoursNNH
N HN
CO2Et CO
1N NaOH THF
BaseHydrolysis
Ru(bpy)2CO3
MeOH, H 2Osep. on Al 2O3N
NNH(C H2)7
Ru
NNH
N HN
CO2H CO
(bpy )2Cl2
NH(C H2)7 N
N
60%
75%
C7PP
RuC7PP
Using UV-visible spectroscopy, we can determine when theC7PP or RuC7PP has been synthesized.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
250
300
350
400
450
500
550
600
Wavelength (nm)
Ab
sorb
ance RuC7PP
C7PP
400 nm
288 nmFor the C7PP, there should be a 1 to 5 ratio between the peaks at 288 and 400 nm. The peak at 288 nm corresponds to bipyridine, and the one at 400 nm represents porphyrin. The spectrum of RuC7PP will have an increase for the 288 nm peak, increasing the aforementioned ratio to 3 to 5, as seen.
Metallation of Heme Cofactor
0
0.2
0.4
0.6
0.8
1
1.2
250
300
350
400
450
500
550
600
650
700
750
Wavelength (nm)
Ab
sorb
anc
e
RuC7FePP400 nm
288 nm
•A UV-visible spectrum was taken to verify the incorporation of iron into the porphyrin of RuC7PP. On the spectrum to the left, it can be seen that there are only two Q bands. This is diagnostic of the metallated porphyrin. The two Q bands are peaks at 550 and 650 nm. Unmetallated porphyrins, eg. RuC7PP and C7PP, have four Q bands.
550 nm650 nm
N
NNH(CH2)7
Ru
NNH
N HN
CO2H CO
(bpy)2Cl2
FeCl2DMF
NN
N N
CO2H CO
Fe
Cl
Reflux, 4 hours sep. on LH20
N
NNH(CH2)7
Ru(bpy)2Cl2
85%
RuC7FePP
Preparation of Apomyoglobin
• The synthesis of apomyoglobin occurs in a three step process:1. Extraction of Native Heme2. Dialysis3. Lyophilization
Myoglobin Apomyoglobin
4oC
The reconstitution of the altered heme is done by mixing it with the apoprotein, and purifying the reformed holoenzyme by chromatography
Molecular Model of Hybrid Mb
Molecular Modelof RuC7FePP myoglobin
Wavelength (nm)300 400 500 600 700
0
0.2
0.4
0.6
0.8
RuC7MbMbRu(bpy)3
2+
ca. 4 uM
Visible spectra of the hybrid Myoglobin
•When reconstituted into apomyoglobin, the UV-visible spectrum resembles what one would expect from a coupling of Ru(bpy)3
2+ and FeIII myoglobin. The soret shifts from 400 nm to 409 nm when reconstituted into protein.
Characterization of the hybridby UV Spectroscopy
LASERLASERBeamsplitterBeamsplitter
sample
O
D
Time
TerminationTermination
Nanosecond Transient Absorption Spectroscopy
MonochromatorMonochromator
Probe LightProbe Light
Photoinduced nanosecond transient absorption
•Using nanosecond transient absorption measurements of the heme soret bands in the hybrid Mb we can follow the fast electron transfer between Rubpy and the FeMb.
•The transient traces illustrate the back electron transfer, kbet, regenerating the FeIII/Ru2+ state is 2 x 107 sec-1
hvk fet
Myoglobin
Fe III
Ru 2+(byp) 3
k -bet
0 1 2 3 4 0 1 2 3 4Time (us) Time (us)
FeIII Soret @ 410 nm
FeII Soret @ 430 nm
h
kbet
kfetFeIII *Ru2+
Ru3+FeII
Ru2+FeIII
Future Work
During the next year we will attempt to reconstitute the RuC7FePP heme cofactor into CcP. In myoglobin the active site is located on the edge of the protein whereas in CcP one of the propionate groups is blocked and the other is recessed from the periphery of the protein. Due to the topographic differences in the active sites, it is probable that we will need to alter the length and characteristics of the pendant arm in order for successful reconstitution.
Other Possible Pendant Groups:-Re(bpy)(CO)3L (L= Cl-, Br-, pyridine)-Methyl viologen
trp 191
trp 51
his 175
his 52
CcP Active Site
N NR'
Acknowledgments
• Farmer Group• Greg Qushair• David Khandabi• Phuong Do
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