Electron paramagnetic resonance metalloenzymes

The possibility that there might be long-range electron transfer between redox-active centers in enzymes was first suspected by biochemists working on the mechanism of action of metalloenzymes such as xanthine oxidase which contain more than one metal-based redox center. In these enzymes electron transfer frequently proceeds rapidly but early spectroscopic measurements, notably those by electron paramagnetic resonance, failed to provide any indication that these centers were close to one another. 

Electron paramagnetic resonance (continued) cobalt-thermolysin complex, 28 334, 335 exchange reactions, 31 106-107 glutamine synthetase, 28 358-364 invisible oxygen species, 31 94-95 metalloenzymes, 28 324, 326 metal particle size distribution, 36 99-100, 104... 

We have mentioned earlier the dissimilarities between the spectral properties of chromophoric metal ions at the active sites of metalloen-zymes and the properties of simple bidentate model complexes of the same metals. Cobalt phosphatase has served well to illustrate such a dissimilarity and, in Figure 9, the data for phosphatase, representative of a cobalt enzyme, are shown again along with those for plastocyanin, a copper enzyme, and ferredoxin, an iron enzyme. Each enzyme spectrum is unusual compared with the simple model complexes shown at the bottom of the figure. More detailed spectral data as well as comparison of other physical properties of metalloenzymes—e.g., electron paramagnetic resonance spectra—with those of model complexes have been summarized previously (10). 

H20. Mass spectrometry confirmed that the product was [ 0]-hydroxyatrazine and thus the reaction was hydrolytic. More recendy, further mechanistic studies have revealed that atrazine chlorohydrolase coordinates ferrous iron, which is essential for its catalytic activity (29). Incubating atrazine chlorohydrolase with the chelators 1,10-phenanthroline or oxalic acid removed the required metal with concomitant loss of activity. Chlorohydrolase activity was restored by the addition of Fe(II), Mn(II), or Co(II) salts. Electron paramagnetic resonance (EPR) and electronic spectroscopic studies provided data consistent with a 1 1 metal to subunit stoichiometry. In total, the data indicate that atrazine chlorohydrolase is a metalloenzyme, making this the first report of a metal-dependent hydrolytic dehalogenase. 

Electron nuclear double resonance (ENDOR) and electron spin-echo envelope modulation (ESEEM) are two of a variety of pulsed EPR techniques that are used to study paramagnetic metal centers in metalloenzymes. The techniques are discussed in Chapter 4 of reference la and will not be discussed in any detail here. The techniques can define electron-nuclear hyperfine interactions too small to be resolved within the natural width of the EPR line. For instance, as a paramagnetic transition metal center in a metalloprotein interacts with magnetic nuclei such as H, H, P, or these... 

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