Structure-function studies of electron transfer complexes in bovine heart mitochondria

Abstract

Various complexes within the bovine heart mitochondrial electron transfer chain were studied. The QPs1 subunit of Succinate: Ubiquinone Reductase was over expressed in Escherichia coli DH5alpha cells as a glutathione S-transferase fusion protein (GST-QPs1) using the expression vector, pGEX/QPs1. Recombinant QPs1 contains little cytochrome b560 heme. However, addition of hemin chloride reconstitutes the spectral characteristics of cytochrome b560. Reconstituted cytochrome b560 in recombinant QPs1 shows a EPR signal at g=2.91. Histidine residues at positions 98 and 120 are responsible for the heme ligation because the H98D or H120D-reconstituted QPs1 fail to restore cytochrome b560 upon addition of hemin chloride.

A purification procedure for NADH dehydrogenase of NADH:Ubiquinone Reductase was developed. This procedure involves acid extraction in the presence of organic solvent followed by ammonium sulfate fractionation, and DEAE column chromatography. Purified NADH dehydrogenase contains 3 protein subunits with apparent molecular weight of 51, 24, and 10 kD. It catalyzes oxidation of NADH by artificial electron acceptor, ferricyanide, with specific activity of 471 micromol Fe(CN)6^3- reduced/min/mg protein. The purified NADH dehydrogenase contains one FMN, five irons, and two EPR detectable iron sulfur clusters, one [4Fe-4S] and one [2Fe-2S]. Preliminary results of crystallization have been discussed.

Using ultra-fast microfluidic mixer and freeze-quenching device, coupled with EPR, the pre-steady state kinetic of ISP reduction by ubiquinol was determined. The complex was mixed with reduced Q0C10Br (QH2) to a final concentration of 50 microM of cytochrome c1, and 333 microM of QH2 in the ultrafast microfluidic mixer and freeze-quenching at various time points, ranging from 100 micros to 5 ms. The first phase reduction of ISP starts between 100 to 200 micros with t1/2 of 250 micros. A similar reduction kinetic is also observed for cytochrome bL with t1/2 micros of 350 micros indicating an almost simultaneously reduction of both ISP and bL. Possible detection of semiquinone radical at Qo site is also discussed.