Oxygen regulation of nitrate transport by diversion of electron flow in Escherichia coli.

Anaerobic nitrate respiration is regulated by oxygen at the level of nitrate transport; however, the mechanism of O2 inhibition is unknown. Potentially, oxygen could inhibit directly by causing conformational changes in the porter system or indirectly through diversion of electron flow from the nitrate reductase complex to oxygen reduction. Inhibition due to electron diversion implies that nitrate reduction is required for nitrate transport. In this regard, nitrate uptake and its regulation by oxygen were studied in mutants of Escherichia coli (strain AN386) deficient in cytochrome d (RG98), cytochrome o (RG101), and a mutant deficient in both cytochrome d and cytochrome o (RG99). Respiratory nitrate uptake in RG99 was highly resistant to the effects of oxygen supporting the indirect mechanism of electron diversion in oxygen regulation. Nitrate transport in RG98 and RG101 is highly sensitive to oxygen; these mutants exhibited 81 and 85% inhibition, respectively, which is similar to inhibition in the wild type. These results indicate that during nitrate respiration, O2 inhibits transport by limiting the supply of electrons to the nitrate reductase complex.

Escherichia coli can respire aerobically at the expense of oxygen or anaerobically using nitrate as a terminal electron acceptor via a membrane-bound nitrate reductase enzyme complex. Oxygen reduction by cytochrome o and d occurs on the inner face of the cytoplasmic membrane as oxygen diffuses through the membrane (1). Nitrate reduction also occurs on the inner aspect of the membrane, but the exact mechanism by which nitrate enters the E. coli cell is unknown (2). Both forms of respiration produce a proton gradient, which is subsequently utilized directly as a source of energy or transformed ATP by the membrane-associated ATPase (3,4). Anaerobic nitrate respiration is regulated at the level of gene transcription (2, 5) or subsequent to gene expression at the level of nitrate transport (6). Anaerobiosis derepresses the synthesis of the proteins involved in nitrate respiration, and the expression of these genes is further enhanced by the presence of nitrate (2). After the induction of the nitrate respiratory system, nitrate reduction is immediately and reversibly regulated by oxygen. It was originally demonstrated in denitrifying organisms (7-9) and later in E. coli (6,10) that oxygen acts by preventing the transport of nitrate to the site * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.  Vol. 265, No. 30, Issue of October 25, pp. 18095-18097, 1990 American Society for Biochemistry and Molecular Biology, Inc.
Printed in U. S. A. of its reduction.
The mechanism of this inhibition is vague and has not been defined.
One possible model for oxygen inhibition of transport is an oxygen-sensitive nitrate porter system. Since the catalytic site of nitrate reductase is located on the inner face of the membrane, a nitrate transport system could be envisioned where oxygen induces a conformational change in the uptake protein thereby preventing nitrate uptake into the cell. In this model, active sulfhydryl groups in the transport protein might be required for nitrate transport.
Thus electrons would be diverted from the nitrate respiratory chain to oxygen respiration, a more energy efficient process.
If the transport of nitrate was dependent on the presence of intracellular nitrite as in a nitrate/nitrite antiport system, a second model can be envisioned which is based on the assumption that nitrate transport is dependent on nitrate reduction. Therefore a diversion of electrons from the nitrate reductase complex would prevent nitrate uptake and alter the redox state of the membrane.
In the presence of oxygen, electrons preferentially flow to cytochrome o or d which reduce oxygen to water; nitrate reductase is therefore starved for electrons, nitrate reduction ceases, and the intracellular level of nitrite required for the transport of nitrate is therefore limited.
The present investigation focuses on the effect of oxygen on nitrate uptake in mutants of E. coli defective in the ability to use oxygen as a terminal electron acceptor for respiration. Therefore, if oxygen regulates anaerobic nitrate respiration through electron diversion to oxygen respiration, this mutant lacking both cytochrome o and d activity should be resistant to changes in oxygen tension during nitrate transport.

AND DISCUSSION
The effect of saturating concentrations of oxygen on nitrate uptake is depicted in Figs. 1 and 2. Under aerobic conditions, nitrate uptake is almost completely inhibited in the wild type (AN386) (Fig. lA). When anaerobic conditions are reestablished, uptake continues once all oxygen has been consumed. This inhibition is both immediate and reversible indicating that nitrate-respiring organisms divert electron flow to a more energy-efficient system of oxygen respiration. In RG99, the mutant deficient in both cytochrome o and d, nitrate uptake is scarcely inhibited when oxygen is introduced into the system and continues at a rate which is at least 60% of the anaerobic rate (Fig. 1B). The effect of oxygen on nitrate uptake in the cytochrome o mutant (RGlOl) and the cytochrome d mutant (RG98) is illustrated in Fig. 2. Both are still quite sensitive to the effects of oxygen. Although cytochrome d is known to exhibit a greater affinity for oxygen since it is predominant under low oxygen tensions (ll), both mutants showed inhibition similar to that which is seen in the wild type AN386.
The effect of oxygen on rates of nitrate uptake in E. coli AN386 and the cytochrome mutants is summarized in Table  I (Table I), suggesting that more than one mechanism for O2 inhibition of transport exists or that the inhibition is mediated by the redox state of the gen in the double mutant RG99 which is unable to utilize membrane, the latter of which has been documented in lactose oxygen as a terminal electron acceptor for respiration. The transport (14). Another interesting note is that the anaerobic effects of oxygen that were observed on nitrate uptake in this rate of nitrate uptake in the cytochrome d and o mutant organism were immediate and completely reversible. Treat-(RG99) consistently shows a greater rate of uptake than the ment of cell suspensions with sulfhydryl inhibitors did not wild type (AN386).
affect the rate of anaerobic nitrate uptake which indirectly One potential mechanism for an oxygen-sensitive porter supports the proposed model of oxygen regulation through might be through essential sulfhydryl groups which may be electron diversion. Furthermore, the degree of oxygen inhibirequired for nitrate transport. In order to test this possibility, tion appears to be regulated by the energy source in the nitrate uptake was measured with concentrations of sulfhy-system. This interesting effect is best illustrated when formate dry1 inhibitors N-ethylmaleimide and iodoacetate ranging is used as an electron donor. Therefore, it appears that oxygen from 10 to 50 pM. However, these inhibitors showed no effect inhibition is probably through the diversion of electrons from on nitrate uptake or oxygen regulation in the wild type or the nitrate reductase to oxygen thereby limiting the reduction of double mutant RG99 (data not shown) suggesting that an nitrate which is required for transport.
oxygen-sensitive nitrate porter system may not be a viable model.

Another
interesting observation is the effect of electron donors on oxygen inhibition of nitrate transport. John's study of nitrate reduction in membrane vesicles of E. coli (13) indicated t,hat the electron source for nitrate reduction had an effect on oxygen inhibition.
We examined this effect in whole cell suspensions to determine if the site at which electrons feed into the electron transport chain affects oxygen regulation of nitrate uptake. Nitrate reduction in whole cells energized by formate is much more resistant to the effects of oxygen than if glucose, succinate, or malate are used (Table  II). This effect may be a result of how and where formate can feed electrons into the electron transport chain. It could also reflect various redox states the membrane might assume when different reduced compounds serve as the primary sources of electrons for respiration.
From these preliminary experiments, it was demonstrated that anaerobic nitrate uptake is much more resistant to oxy-