2. Introduction Our 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

3. Synthesis of Bpy Pendant Arm Using this method, we can systematically vary the length and composition of the pendant arm. Synthesis of Protoporphyrin IX Derivative This protects one proprionate which will be nessasary to help stabilize our reconstituted protein.

4. The Amide Coupling Reaction Using UV-visible spectroscopy, we can determine when the C 7 PP or RuC 7 PP has been synthesized. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 250300 350400 450500 550 600 Wavelength (nm) Absorbance RuC 7 PP C 7 PP 400 nm 288 nm For the C 7 PP, 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 RuC 7 PP will have an increase for the 288 nm peak, increasing the aforementioned ratio to 3 to 5, as seen.

5. Metallation of Heme Cofactor 0 0.2 0.4 0.6 0.8 1 1.2 25 0 30 0 35 0 40 0 45 0 50 0 55 0 60 0 65 0 70 0 75 0 Wavelength (nm) Absorbanc e RuC 7 FePP 400 nm 288 nm A UV-visible spectrum was taken to verify the incorporation of iron into the porphyrin of RuC 7 PP. 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. RuC 7 PP and C 7 PP, have four Q bands. 550 nm 650 nm

6. Preparation of Apomyoglobin The synthesis of apomyoglobin occurs in a three step process: 1. Extraction of Native Heme 2. Dialysis 3. Lyophilization Myoglobin Apomyoglobin 4oC4oC The reconstitution of the altered heme is done by mixing it with the apoprotein, and purifying the reformed holoenzyme by chromatography

7. Molecular Model of Hybrid Mb Molecular Model of RuC 7 FePP myoglobin

8. Wavelength (nm) 300400500600700 0 0.2 0.4 0.6 0.8 RuC7Mb Mb Ru(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 Fe III myoglobin. The soret shifts from 400 nm to 409 nm when reconstituted into protein. Characterization of the hybrid by UV Spectroscopy

9. LASERBeamsplitter sample  OD Time Termination Nanosecond Transient Absorption Spectroscopy Monochromator Probe Light

10. 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, k bet, regenerating the Fe III /Ru 2+ state is 2 x 10 7 sec -1 h v k f et Myoglobin Fe III Ru 2+ (byp) 3 k - b et 01234 01234 Time (us) Fe III Soret @ 410 nm Fe II Soret @ 430 nm

11. Future Work During the next year we will attempt to reconstitute the RuC 7 FePP 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

12. Acknowledgments Farmer Group Greg Qushair David Khandabi Phuong Do