The Distribution of Redox Equivalents in the Anaerobic Respiratory Chain of Paracoccus denitrificans

Abstract

The branching of the electron flow to individual teminal acceptors NO3−, NO2− and N2O was investigated in denitrifying cells of Paracoccus denitrificans. It was found that the extent of electron flow to nitrate depends on the activity of electron flow through the terminal part of the respiratory chain comprising cytochrome c. NO2− and N2O either endogenously formed or exogenously added decreased the rate of nitrate reduction by oxidizing the respiratory chain via nitrite reductase and nitrous oxide reductase. The inhibitory effect of both intermediates of denitrification was released by antimycin. The rate of the first reaction of the sequence NO3−→ NO2−→ N2O → N2 is in the type of cells used under feedback redox control exerted by concentration of intermediates. This regulation can be designated as ‘inhibition by product via respiratory chain’. The reaction rate of nitrate denitrification could also be influenced by changing the portion of electron flow from substrate to cytochrome c by artificial electron acceptors and donors. By varying the concentration of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine in the presence of ascorbate, the desired redox state of cytochrome c and consequently various rates of nitrate reductase activity could be established. The basis of the enhancing effect of dithionite on the rate of nitrate reduction could also be explained by its functioning as an electron donor for the terminal part of respiratory chain. The quantitative evalution of the extent of electron flow revealed that both branches of the respiratory chain (to nitrate reductase and to cytochrome c) compete for a constant limited flow of redox equivalents supplied from dehydrogenases. A strong evidence exists that the same mechanism is implied in the inhibition of nitrate reduction brought about by oxygen.

The function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors

“…by means of a technique used before [4]. The results of these experiments given in Fig.6 are analogous to those presented in Fig.…”

“…The redox state of cytochrome c at different time intervals was followed by registering the difference spectra of cells suspended in 3 ml of the basic medium using the spectrophotometer Cary 118 C, as described before [4].…”

“…Paracoccus denitrificans (NCIB 8944) was grown anaerobically in a medium as described earlier [4] except the fact that glucose was substituted with 50 mM sodium succinate. The cells were harvested in an early stationary phase of growth, washed with 50 mM sodium phosphate of pH 7.3 and suspended in the same buffer.…”

“…Thus, the addition of oxygen results in an immediate block of nitrate consumption [3]; similarly, the reduction of nitrate is partly inhibited even by the utilization of endogenously originating or externally added NO; and N20 [4]. It has been proved by several independent methods that the above phenomena depend on the redox state of the components of the respiratory chain in the branching sites [4-61 and that they can be described as the competition of terminal branches for a constant limited influx of redox equivalents from dehydrogenases [4].There are, however, experimental results indicating that this simple description of the control of electron fluxes to the individual terminal acceptors need not be sufficient. It has actually been found that membrane vesicles [3] and/or cells treated with the detergent Triton X-100 [7] are capable of reducing nitrate even in the presence of oxygen.…”

“…In the presence of more than one acceptor a preferential reduction of one of them can be observed; simultaneously the reduction of other acceptors is inhibited. Thus, the addition of oxygen results in an immediate block of nitrate consumption [3]; similarly, the reduction of nitrate is partly inhibited even by the utilization of endogenously originating or externally added NO; and N20 [4]. It has been proved by several independent methods that the above phenomena depend on the redox state of the components of the respiratory chain in the branching sites [4-61 and that they can be described as the competition of terminal branches for a constant limited influx of redox equivalents from dehydrogenases [4].…”