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The kinetics of NADH oxidation by complex I of isolated plant mitochondria
Author(s) -
Sooie Kathleen L.,
Dry Ian B.,
James Alan T.,
Wiskich Joseph T.
Publication year - 1990
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1990.tb04377.x
Subject(s) - submitochondrial particle , rotenone , nadh dehydrogenase , chemistry , ferricyanide , reductase , oxygen , kinetics , electron transport chain , mitochondrion , biochemistry , stereochemistry , enzyme , organic chemistry , physics , protein subunit , quantum mechanics , gene
The oxidation of matrix NADH in the presence and absence of rotenone was investigated in submitochondrial particles prepared from purified beetroot ( Beta vulgaris L.) mitochondria. The submitochondrial particles oxidised NADH using oxygen and artificial electron acceptors such as ferricyanide (FeCN) and short‐chain analogues of ubiquinone(UQ)‐10, although the NADH‐FeCN reductase activity was not inhibited by rotenone. NADH‐oxygen reductase activity in the presence and absence of rotenone displayed different affinities for NADH (145 ± 37 and 24 ± 9 μ M , respectively). However, in the presence of 0.15 m M UQ‐1 where any contribution from non‐specific sites of UQ‐reduction was minimal, the rotenone‐insensitive oxygen uptake was stimulated dramatically and the K m (NADH) decreased from 167 ± 55 μ M to 11 ± 1 μ M ; a value close to that determined for the total oxygen uptake which itself was virtually unaffected by the addition of UO‐1 [K m (NADH) of 13 ± 3 μ M ). The similar affinity of NADH‐oxygen reductase for NADH when UQ‐1 was present in both the presence and absence of rotenone, suggested that there may be only one NADH binding site involved in the two activities. A quantitative two‐stage model for Complex I is postulated with one NADH binding site and two sites of UQ‐reduction (one of which is insensitive to rotenone) with a common intermediate ‘P’ whose level of reduction can influence the NADH binding site. The poor affinity that rotenone‐insensitive NADH‐oxygen reductase activity displayed for NADH results from a limitation on the interaction of its UQ‐reduction site with UQ‐10 in the membrane; possibly from a low concentration of UQ‐10 around this site or from steric hindrance restricting the access of UQ‐10 to this reduction site.