Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate

Abstract

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.

Electron spin relaxation enhancement measurements of interspin distances in human, porcine, and Rhodobacter electron transfer flavoprotein–ubiquinone oxidoreductase (ETF–QO)

“…An algorithm predicts ≤ 0.7 root mean square deviation/Å in the cores of the three proteins based on the high degree of sequence identity [6]. Consistent with the similarity in sequences, EPR g-values are comparable for the [4Fe-4S] + in mammalian ETF-QOs [1,3,7] as are the redox potentials of porcine and Rhodobacter ETF-QO [5]. Thus, it is probable that the Rhodobacter, human, and porcine ETFQOs have similar structures.…”

“…Porcine liver ETF-QO was purified as described by Watmough and co-workers [30]. Human ETF-QO was expressed from a baculovirus vector, expressed in sf9 cells, and purified by the method of Simkovic et al [3].…”

“…The ETF-QO from Rhodobacter sphaeroides was expressed in Escherichia coli C43 [31] and purified by the method reported in Usselman et al [32]. The concentrations of the three proteins were determined spectrophotometrically using ε 430nm = 2.4×10 4 M -1 cm -1 [3,5,9]. The purified proteins do not contain ubiquinone so throughout this manuscript quinone and semiquinone refer to the FAD moiety.…”

“…Ubiquinone was not present in the proteins isolated for this study. Porcine and human ETF-QO have 98% sequence identity, whereas human and Rhodobacter sphaeroides ETF-QO ETF-QO have 67% sequence identity [4,5]. An algorithm predicts ≤ 0.7 root mean square deviation/Å in the cores of the three proteins based on the high degree of sequence identity [6].…”

“…Because the two redox potentials for the FAD and the potential for the [4Fe-4S] are so similar, this enzymatic reduced state is a redox equilibrium involving partial reduction of the [4Fe-4S] and of the FAD [3,5,8,9]. A stronger reductant, such as dithionite, is required to reduce the protein to a nominally "three-electron reduced" state [3,5,8,9].…”