PubMed · 7752937
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G W Brudvig. 1995. Electron paramagnetic resonance spectroscopy.. https://doi.org/10.1016/0076-6879(95)46024-1
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The 2-oxo-1,2-dihydroquinoline 8-monooxygenase from Pseudomonas putida 86 comprises two components with four redox active sites necessary for activity. We present an EPR characterization of the iron-sulfur centres in the purified reductase and oxygenase component of this novel enzyme system. The oxygenase component was identified as a Rieske [2Fe2S] protein on the basis of its characteristic EPR spectrum with gz,y,x = 2.01, 1.91, 1.76 and gav = 1.893. The reductase component, an iron-sulfur flavoprotein, contained a [2Fe2S] cluster with gz,y,x = 2.03, 1.94, 1.89 and the average g-value (gav) of 1.953, typical of a ferredoxin-type centre. In redox titrations at pH 7, the midpoint potentials were determined to be -180 mV +/- 30 mV and -100 mV +/- 10 mV for the reductase and oxygenase component, respectively. A detailed comparison to other multicomponent enzyme systems is presented pointing out the EPR and redox properties of the FeS centres involved.
The mobility of spin-labeled lipids has been studied in dioleoyl and dimyristoyl phosphatidylglycerol bilayers and in their complexes with alpha-lactalbumin at pH 4.0, by using electron spin resonance (ESR) spectroscopy. The ESR spectra of phosphatidylglycerol spin-labeled at position 5 of the sn-2 chain indicate that association of alpha-lactalbumin with dimyristoyl phosphatidylglycerol bilayers increases the chain mobility at temperatures in the lipid gel phase, restricts the chain mobility at temperatures corresponding to the lipid fluid phase, and abolishes the cooperative lipid chain-melting transition. The ESR spectra of phosphatidylglycerols spin-labeled at eight different positions in the sn-2 chain show that binding of alpha-lactalbumin to dioleoyl phosphatidylglycerol bilayers at pH 4.0 causes a motional restriction throughout the full length of the lipid acyl chain. For phosphatidylglycerols spin-labeled at the terminal methyl end of the chains, a population of motionally restricted lipids that directly contacts membrane penetrant portions of the protein is detected. This population corresponds to 6.3 +/- 0.7 lipids/alpha-lactalbumin at saturation binding, and the high degree of motional restriction (maximum hyperfine splitting approximately 60 G) suggests that the protein may traverse the lipid bilayer. A small selectivity of phosphatidylglycerol over zwitterionic phospholipids for interaction with alpha-lactalbumin is found at subsaturating levels of binding at pH 4.0. Binding of alpha-lactalbumin also strongly restricts the motion of lipids spin-labeled in the polar head group region. These results are of direct relevance to the insertion and translocation of a protein in the molten globule state across lipid membranes.
We have found that the only high redox potential electron transfer component in the soluble fraction of Rubrivivax gelatinosus TG-9 is a high-potential iron-sulfur protein (HiPIP). We demonstrated the participation of this HiPIP in the photoinduced electron transfer both in vivo and in vitro. First, the addition of HiPIP to purified membranes enhanced the rate of re-reduction of the photooxidized reaction center. Second, the photooxidation of HiPIP was observed in intact cells of Ru. gelatinosus TG-9 under anaerobic conditions by EPR and absorption spectroscopies. Analysis of flash-induced absorption changes showed that the equilibration of positive equivalents between the reaction center and HiPIP occurs in less than 1 ms after flash excitation. The complete re-reduction of the photooxidized reaction center is achieved in tens of milliseconds. The turnover of a cyt bc1 is also involved in this reaction, as shown by a slow electrogenic phase of the membrane potential linked to this process.