Restoration of NADH Oxidation with Menaquinones and Menaquinone Analogues in Membrane Vesicles from the Menaquinone‐Deficient Bacillus subtilis aroD

Abstract

Membrane vesicles from the menaquinone-deficient Bacillus subtilis aroD oxidize NADH at a low rate. NADH oxidation can be restored by the addition of slightly water-soluble menaquinone and ubiquinone analogues up to saturation levels. These saturation levels differ for the different quinone analogues tested from 95 (1,4-benzoquinone) to 5316 (5-hydroxy-1,4-naphthoquinone, juglon) nmol NADH x min-' x mg membrane protein-' N A D H oxidation in membrane vesicles from B. subtilis aroD restored with water-soluble quinone analogues supplies the energy for L-glutamate uptake. Like N A D H oxidation the initial rate of L-glutamate transport increases up to saturation levels. The highest initial rates of L-glutamate uptake are observed after restoration with quinone analogues with a relatively low standard redox potential.Functional reconstitution with natural, water-insoluble, quinones can be achieved effectively by mixing quinone-containing liposomes with membrane vesicles from B. subtilis aroD and subsequent freezing of the mixture in liquid nitrogen. The rate of N A D H oxidation increased with the amount of menaquinone incorporated in the vesicles up to saturation levels. NADH oxidation via these menaquinones also supplies the energy for L-glutamate uptake. The highest uptake rates can be obtained with menaquinone-1 and menaquinone-2.On the basis of efficiencies (mol NADH oxidized/mol L-glutamate transported) menaquinones and menaquinone analogues can be divided in two classes.To class 1 belong the menaquinone analogues, menaquinone-5 and menaquinone-8. These compounds restore NADH oxidation with low levels of energy transduction. Efficiencies are observed which are comparable with the efficiency observed in membrane vesicles from B. subtilis W23 (120-140) which contain the natural menaquinone-7.To class 2 belong menaquinone-I and menaquinone-2, which restore NADH oxidation with high levels of energy transduction. Efficiencies are observed which are in the same range as observed with phenazine methosulphate (9 -13).A model is proposed in which class 1 compounds feed in electrons from the outside from NADH to the Q-cycle of the respiratory chain. Class 2 compounds donate electrons to the respiratory chain after cytochrome c and before cytochrome a-601.Secondary transport of many solutes in membrane vesicles of Bacillus subtilis can be energized by electron transfer in the respiratory chain [l -51. The best electron donors for energization are reduced /I-nicotinamide-adenine-dinucleotide (NADH) and ascorbate/phenazine methosulphate [5 -71. A mutant has been isolated by Farrand and Taber [8,9] which is deficient in the synthesis of menaquinone, B. subtilis aroD (RB163). Membrane vesicles of this mutant oxidize NADH at a very low rate and NADH does not function as an energy source for solute transport [6,10].In order to obtain information about the structural requirements and the functional properties of menaquinones in the respiratory chain such a mutant is of particular interest. Incorporation of menaquinones in membr...

Role of menaquinone in inactivation and activation of the Bacillus cereus forespore respiratory system

“…This result is in agreement with previous conclusions (6) on the role of menaquinone as the rate-limiting step for NADH oxidase in dormant spores. Bergsma et al (3) reported that menadione is a more efficient electron transporter in B. subtilis than endogenous menaquinone is; ap- 0) (Fig. 2).…”