PubMed · 9102295
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W Rosenthal, M Brumhard. 1997-02-28. [Coenzyme Q10].. https://pubmed.ncbi.nlm.nih.gov/9102295/
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We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes.
We measured the lateral diffusion of different coenzyme Q homologues and analogues in model lipid vesicles using the fluorescence collisional quenching technique with pyrene derivatives and found diffusion coefficients in the range of 10(-6) cm2/s. Theoretical diffusion coefficients for these highly hydrophobic components were calculated according to the free volume theory. An important parameter in the free volume theory is the relative dimension between diffusant and solvent: a molecular dynamics computer simulation of the coenzymes yielded their most probable geometries and volumes and revealed surprisingly similar sizes of the short and long homologues, due to a folded structure of the isoprenoid chain in the latter, with a length for coenzyme Q10 of 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s, in good agreement with those found experimentally.