We have succeeded in designing and constructing de novo synthetic flavo-hemo-proteins that exhibit light activated intraprotein electron transfer. We use our traditional four-helix bundle framework to covalently link a bromoflavin to an interior cysteine very close to the geometric center of the bundle. In the same bundle, interior histidines a positioned to form a bis-His ligation of added heme toward either end of the bundle interior. We have shown that illumination of the blue flavin absorption band creates an excited state which can extract a pair of electrons from a convenient nitrogenous base donor (such as EDTA) to create a reduced flavin that spontaneously transfers electrons to nearby bis-histidine ligated heme. We are presently using the RLBL nanosecond dye laser to capture the spectra of the excited flavin state, the subsequent reduced flavin, and to provide an initial description of the time course of intraprotein electron transfer from the reduced fla vin to the heme. It is quite possible that there will be a heterogeneity of the time course corresponding to a distribution of flavin/heme distances for different conformations of the redox peptide. We expect a competition between flavin excited state quenching by energy transfer to the heme and electron transfer between the flavin and the heme. We will be flowing the synthetic flavo-hemo-protein plus EDTA solution through a cell to compare the spectra of regions with and without the blue pump pulse. We can easily regenerate our material by exposing the solution to molecular oxygen, which rapidly oxidizes the heme component. In addition, we are investigating the viability of using a tryptophan moiety in this electron transfer scheme. The tryptophan residue will be pumped with the fourth harmonic of the Nd:YAG (266 nm).

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001348-19
Application #
6328042
Study Section
Project Start
2000-08-01
Project End
2001-07-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
19
Fiscal Year
2000
Total Cost
$8,177
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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