Yumiko Nakashima

Bovine Heart NADH―Ubiquinone Oxidoreductase Contains One Molecule of Ubiquinone with Ten Isoprene Units as One of the Cofactors

NADH-ubiquinone oxidoreductase (Complex I) is located at the entrance of the mitochondrial electron transfer chain and transfers electrons from NADH to ubiquinone with 10 isoprene units (Q(10)) coupled with proton pumping. The composition of Complex I, the largest and most complex proton pump in the mitochondrial electron transfer system, especially the contents of Q(10) and phospholipids, has not been well established. An improved purification method including solubilization of mitochondrial membrane with deoxycholate followed by sucrose gradient centrifugation and anion-exchange column chromatography provided reproducibly a heme-free preparation containing 1 Q(10), 70 phosphorus atoms of phospholipids, 1 zinc ion, 1 FMN, 30 inorganic sulfur ions, and 30 iron atoms as the intrinsic constituents. The rotenone-sensitive enzymatic activity of the Complex I preparation was comparable to that of Complex I in the mitochondrial membrane. It has been proposed that Complex I has two Q(10) binding sites, one involved in the proton pump and the other functioning as a converter between one and two electron transfer pathways [Ohnishi, T., Johnson, J. J. E., Yano, T., LoBrutto, R., and Widger, R. W. (2005) FEBS Lett. 579, 500-506]. The existence of one molecule of Q(10) in the fully oxidized Complex I suggests that the affinity of Q(10) to one of the two Q(10) sites is greatly dependent on the oxidation state and/or the membrane potential and that the Q(10) in the present preparation functions as the converter of the electron transfer pathways which should be present in any oxidation state.

Steady-State Kinetics of NADH:coenzyme Q Oxidoreductase Isolated from Bovine Heart Mitochondria

Steady-state kinetics of the bovine heart NADH:coenzyme Q oxidoreductase reaction were analyzed in the presence of various concentrations of NADH and coenzyme Q with one isoprenoid unit (Q1). Product inhibitions by NAD+ and reduced coenzyme Q1 were also determined. These results show an ordered sequential mechanism in which the order of substrate binding and product release is Q1–NADH–NAD+–Q1H2. It has been widely accepted that the NADH binding site is likely to be on the top of a large extramembrane portion protruding to the matrix space while the Q1 binding site is near the transmembrane moiety. The rigorous controls for substrate binding and product release are indicative of a strong, long range interaction between NADH and Q1 binding sites.

Effect of the Side Chain Structure of Coenzyme Q on the Steady State Kinetics of Bovine Heart NADH: Coenzyme Q Oxidoreductase

Steady state kinetics of bovine heart NADH: coenzyme Q oxidoreductase using coenzyme Q with two isoprenoid unit (Q2) or with a decyl group (DQ) show an ordered sequential mechanism in which the order of substrate binding and product release is NADH-Q2 (DQ) -Q2H2 (DQH2)-NAD+ in contrast to the order determined using Q1 (Q1-NADH-NAD+-Q1H2) (Nakashima et al., J. Bioenerg. Biomembr.34, 11–19, 2002). The effect of the side chain structure of coenzyme Q suggests that NADH binding to the enzyme results in a conformational change, in the coenzyme Q binding site, which enables the site to accept coenzyme Q with a side chain significantly larger than one isoprenoid unit. The side chains of Q2 and DQ bound to the enzyme induce a conformational change in the binding site to stabilize the substrate binding, while the side chain of Q1 (one isoprenoid unit) is too short to induce the conformational change.

The Second Coenzyme Q1 Binding Site of Bovine Heart NADH: Coenzyme Q Oxidoreductase

The rotenone sensitivity of bovine heart NADH: coenzyme Q oxidoreductase (Complex I) depends significantly on coenzyme Q1 concentration. The rotenone-insensitive Complex I reaction in Q1 concentration range above 300 μM indicates an ordered sequential mechanism with Q1 and reduced Q1 (Q1H2) as the initial substrate to bind to the enzyme and the last product to be released from the enzyme product complex, respectively. This is the case in the rotenone-sensitive reaction although both Km and Vmax values of the rotenone-insensitive reaction for Q1 are significantly higher than those of the rotenone-sensitive reaction (Nakashima et al., 2002, J. Bioenerg. Biomemb.34, 11–19). This rigorous control mechanism between the nucleotide and ubiquinone binding sites strongly suggests that the rotenone-insensitive reaction is also physiologically relevant.

Two-dimensional crystallization of intact F-ATP synthase isolated from bovine heart mitochondria.

Mitochondrial F-ATP synthase produces the majority of ATP for cellular functions requiring free energy. The structural basis for proton motive force-driven rotational catalysis of ATP formation in the holoenzyme remains to be determined. Here, the purification and two-dimensional crystallization of bovine heart mitochondrial F-ATP synthase are reported. Two-dimensional crystals of up to 1 µm in size were grown by dialysis-mediated detergent removal from a mixture of decylmaltoside-solubilized 1,2-dimyristoyl-sn-glycero-3-phosphocholine and F-ATP synthase against a detergent-free buffer. A projection map calculated from an electron micrograph of a negatively stained two-dimensional crystal revealed unit-cell parameters of a = 185.0, b = 170.3 Å, γ = 92.5°.

Effect of pH on the Steady State Kinetics of Bovine Heart NADH: Coenzyme Q Oxidoreductase

Complete initial steady state kinetics of NADH-decylubiquinone (DQ) oxidoreductase reaction between pH 6.5 and 9.0 show an ordered sequential mechanism in which the order of substrate bindings and product releases is NADH-DQ–DQH2-NAD+. NADH binding to the free enzyme is accelerated by protonation of an amino acid (possibly a histidine) residue. The NADH release is negligibly slow under the turnover conditions. The rate of DQ binding to the NADH-bound enzyme and the maximal rate at the saturating concentrations of the two substrates, which is determined by the rates of DQH2 formation in the active site and releases of DQH2 and NAD+ from the enzyme, are insensitive to pH, in contrast to clear pH dependencies of the maximal rates of cytochrome c oxidase and cytochrome bc1 complex. Physiological significances of these results are discussed.