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