Applications of Pulse Radiolysis to Protein Chemistry

Abstract

Since its introduction, pulse radiolysis has been an important technique for examining the properties of organic and inorganic radicals, and for enumerating those reactions responsible for cellular damage by ionizing radiation. Biochemists, and biophysicists outside the area of radiation biology appear, perhaps for historical reasons, to have an incomplete appreciation of the technique's potential. Protein chemists in particular, have been only dimly aware of the numerous reports of, and the significant results obtained from pulse radiolysis studies of proteins. Our purpose here is to bring some of these results together in order to emphasize the power and usefulness of pulse radiolysis experiments both for elucidating enzyme reaction mechanisms, and for gaining information on the structure of proteins in aqueous solutions. Reviews containing related, or in part the same material to be covered here have appeared previously; for example, Land (1970), Adams et al. (1972a), Shafferman & Stein (1975), Adams & Wardman (1977). This review updates these earlier works, but more importantly approaches the topic of protein pulse radiolysis with a different emphasis.

Formation and spectra of flavin radicals in the presence of β-mercaptoethanol at pH 9 and 10

“…Both the FAD and LFl the spectra of the .FlH radicals formed via reactions [8] and [-101 were the same in the presence and absence of RSH at a concentration of M. It is conceivable that interactions causing spectral changes might occur at higher concentrations. However, since -C02-reacts with RSH with a rate constant of about 1 X 10' M -' s-' Attention is now focused on experiments performed at pH 10, where the flavin radicals should be fully ionized and the sulphydryl about 50 percent ionized, and pH 9, where the former is still largely ionized and the latter about 10 percent ionized.…”

“…1 is the absorption spectrum of the FAD radical produced in reactions [5]- [8] It should be noted that the peak at 370 nm was reproducible to within about 5 percent between experiments, but absorbances in the wavelength range 410 to 480 nm tended to scatter more, both in N20-and argon-saturated solutions. That feature has been illustrated by the data points shown…”

“…Apart from their intrinsic interest to the field of sulphur chemistry, it has been recognised that disulphide anions may be involved in electron transport (4,8). Flavin molecules are important prosthetic groups for a large variety of redox enzymes (9,10) and in the specific cases of glutathione reductase, lipoamide dehydrogenase, and thioredoxin reductase protein-bound disulphides are involved in the two-electron-transfer pathways, which in the direction of substrate oxidation can be represented as (11)(12)(13):…”

First Crystal Structure of a Fungal High-redox Potential Dye-decolorizing Peroxidase

“…Thus, in such a case, a novel protein-based substrate interaction site is introduced. This requires a long-range electron transfer (LRET) pathway (42) from the porphyrin ring to an appropriate redox active amino acid residue at the surface of the enzyme. An exposed oxidation site has been demonstrated extensively in a related heme enzyme viz.…”

Long-range intramolecular electron transfer in azurins.

“…1 A and C). While at 625 nm the Cu(II) site has its absorption maximum with E = 5700 M-1-cm-1 (20), it hardly absorbs at 410 nm, where the disulfide radical ion, formed upon reduction by CO-, has an E = 10,000 M-1'cm-1 (21). Both reactions were found to be first order in protein and in CO-radical concentrations.…”