Cytochrome bd is a respiratory oxidase in Escherichia coli and many other bacteria. It contains cytochromes b5,*, b,,, and d as redox centres, and is thus unrelated to the haem-copper super-family of terminal oxidases. The apparent affinities (K,) for oxygen uptake by respiring cells and membranes from a mutant lacking the alternative oxidase cytochrome bo' were determined by deoxygenation of oxyleghaemoglobin as a sensitive reporter of dissolved oxygen concentration. Respiration rates were maximal a t oxygen concentrations of 25-50 nM, but the kinetics were complex and indicative of substrate (i.e. oxygen) inhibition. K, values were in the range 3-8 nM (the lowest recorded for a respiratory oxidase), and Ki values between 0.5 and 1-8 pM were obtained. Low temperature photodissociation of anoxic, COligated membranes confirmed the absence of cytochrome bo' and revealed a high-spin 6-type cytochrome identified as cytochrome b,,, of the cytochrome bd complex. Photodissociation in the presence of oxygen revealed binding of a ligand (presumably oxygen) to cytochrome b,,, a t a rate much greater than that of CO binding, and formation of the oxygenated form of cytochrome d. The results confirm that both high-spin haems in the cytochrome bd complex bind CO and demonstrate that oxygen can also react with both haems. Substrate inhibition of oxidase activity, in addition to transcriptional regulation of oxidase synthesis, may play a crucial role in the regulation of partitioning of electron flux between the cytochrome bd-and bo'-terminated respiratory pathways.

Azotobacter vinelandii is an obligately aerobic diazotrophic bacterium with two known terminal oxidases of the cytochrome o-and bd-types. The latter is required for respiratory protection of the oxygen-labile nitrogenase during aerotolerant nitrogen fixation. The apparent affinities (K,) for oxygen uptake by A. vinelandii cells and membranes respiring DL-malate have been determined by using the deoxygenation of oxyleghaemoglobin or oxymyoglobin as sensitive reporters of dissolved oxygen concentration. Dualwavelength spectrophotometery allowed continuous recording of oxygen consumption over the range 0-003-10 pM, and revealed three distinct affinities for oxygen in a wild-type strain. The kinetic properties of each oxidase were distinguished by the use of two mutants, one lacking and one over-producing the cytochrome bd-type oxidase. The deoxygenation kinetics of oxyleghaemoglobin revealed a high affinity oxidase in all three strains with Km values for membrane preparations of 0013-0919 pM. In strains having the cytochrome bd-type oxidase, the Km values measured with intact cells were approximately fourfold higher than in membranes. These results suggest a barrier to the transfer of oxygen to the high affinity component by cytochrome bd, perhaps due to very fast oxygen binding or scavenging by cytochrome d , or to the location of the oxygen-consuming sites of these oxidases on different faces of the membrane. The deoxygenation kinetics of oxymyoglobin revealed the presence of two components with mean Km values of about 033 and 4.5 pM. The 4.5 pM component is attributed to the cytochrome bd-type oxidase because it was lacking in intact cells and membranes of the cytochrome bd-deficient mutant strain. The other two components (one with a mean Km value of about 033 pM and the highest affinity activity) could not be assigned to particular oxidase(s). The results are interpreted in relation to the physiological role of the cytochrome bdterminated branch of the respiratory chain and the much higher affinities for oxygen reported for the cytochrome bd-type oxidase in other bacteria.

Apparent oxygen affinities forEscherichia coli contains two terminal quinol oxidases (21,22,28). One of these, cytochrome boЈ, contains the ligandbinding heme O and a second low-spin heme of either the B or O type (29). This oxidase has also been called cytochrome b 562 o (16), bo 3 , and oo 3 (29), but the boЈ nomenclature is in accordance with IUB recommendations (25). Cytochrome boЈ is a member of the heme-copper superfamily of terminal oxidases (7) and is a proton pump (28). The alternative oxidase, cytochrome bd, is structurally unrelated to this family (13) and appears not to pump protons (28). Cytochome boЈ is synthesized under conditions of high aeration (8, 15), whereas maximal synthesis of cytochrome bd occurs microaerobically (12). Apparently consistent with these patterns of regulation are results obtained from studies of whole cells (30) and purified oxidases (16) suggesting that cytochrome bd has a high oxygen affinity, whereas cytochrome boЈ has a much lower affinity. However, these measurements, and a recent claim that E. coli cytochrome bd has a surprisingly low affinity (17), have relied upon polarographic methods in which the sensitivity at low oxygen tensions is limited by the unstirred layer at the electrode membrane (18). Recognizing the physiological significance of the disparate oxygen affinities yet the absence of reliable data on their values, we have measured the K m values for oxygen using a strain containing only cytochrome boЈ by a method that allows highly sensitive and continuous monitoring of oxygen consumption (4).E. coli UNF3502 (14) was constructed by transduction of UNF3501 (trp his rpsE) with bacteriophage P1 grown on GO103, which carries a kanamycin resistance cassette close to the partially deleted cydAB locus. Strain AN2342 is regarded as wild type (27), has a different genetic background, and was used to assess the generality of the data obtained with UNF3502. Cells were grown at 37ЊC, with shaking (200 rpm), in five 2-liter baffled flasks (Belco Biotechnology, Vineland, N.J.), each containing 400 ml of Luria-Bertani medium (LB) (19) supplemented with 0.2% (wt/vol) glucose and (for UNF3502) kanamycin (50 g/ml). Cultures were grown to midexponential phase, harvested, and washed, and a portion of the cell paste (approximately 1 g) was resuspended in twice its volume of 25 mM phosphate buffer (pH 7.5 ml). The suspension was kept at 4ЊC and used for K m determinations within 24 h. Remaining cells were suspended in 50 ml of 50 mM PIPES [piperazine-N,NЈ-bis(2-ethanesulfonic acid)] buffer (pH 6.5), containing 8 mM magnesium acetate and a few grains of DNase (Sigma), and stored at Ϫ70ЊC for membrane preparation.We determined the apparent affinity for oxygen uptake by intact cells and membranes as previously described (10). In brief, oxygenated soybean leghemoglobin or myoglobin was diluted to 10 to 15 M in phosphate buffer (25 mM, pH 7.0) containing 1 mM EDTA, previously sparged with 1% oxygen in argon. Globin solutions were kept under argon plus 1% oxygen and used within 6 h. D...

The influence of the rate of 0, supply to batch cultures on the contents of cytochromes bd and '0' in NHt-grown Azotobacter vinelsndii has been investigated. Difference spectra at room temperature (reduced + CO minus reduced) were recorded for whole cells of a wild-type strain and mutants which either lacked or over-produced the cytochrome bd-type terminal oxidase encoded by cydAB. A Tn5-BZO insertion in cydB in the former mutant also provided a means of monitoring cydAB gene expression from measurements of p-galactosidase activity. The content of cytochrome d in the wild-type, and the expression of cydAB-lacZ, in the mutant, increased as the 0, supply was raised, suggesting that 0 , regulates cydAB expression even in the absence of diazotrophy. In a strain carrying a mutation in cydl?, a regulatory gene upstream of cydAB. and which over-produces cytochrome bd# the responses to 0, supply during growth at different 0, supply rates were reversed. Changes in the content of a haemoprotein detectable in low temperature photodissociation spectra, and attributed to cytochrome b,,, -the high-spin cytochrome b component of the cytochrome bd complex -followed the changes in cytochrome d levels. CO difference spectra of both the wild-type strain and the cytochrome bd-deficient mutant revealed a haemoprotein with spectral characteristics similar to cytochrome 0 . the levels of which increased as the 0 , supply was raised. These results are discussed with reference to previous reports of cytochrome changes in cells grown under N,-f ixing conditions. (Poole, 1983;D'mello et al., 1994b). However, there is a remarkable difference between the E. coli and A. vinelandii oxidases with respect to regulation of synthesis. The E. coli cytochrome bd-type oxidase is synthesized maximally microaerobically (Cotter et al., 1990;Fu et al., 1991;Tseng et al., 1996). Expression of the cydAB operon comprising the oxidase structural genes is affected by Fnr and ArcA/ArcB, although dissection of the direct or indirect roles of these regulators requires further study (Gennis & Stewart, 1996). Cytochrome 6d levels are also elevated at low oxygen tensions in the diazotroph Klebsiella pneumoniae (Smith et al., 1990). In contrast, the level of cytochrome bd in N,-fixing A. vinelandii oxidase is greatest under high aeration (Drozd & Postgate, 1970;Jones et al., 1973;Haaker & Veeger, 1976), although one report describes the reverse situation (Ackrell & Jones, 1971). Accompanying the increase in the cytochrome bd level is a loss of energy conservation at ' Site I' (Jones et al., 1973). Consistent with the majority of these observations is the finding that cytochrome bd in A. vinelandii has a low affinity for 0, (apparent K , = 4.5 pM; D'mello et al., 1994a), yet exhibits a higher velocity for electron transport than does the alternative oxidase 'cytochrome 0 ' (Hoffman et al., 1979;D'mello et al., 1994a). In marked contrast, the oxidase in E. coli has the highest affinity yet recorded ( K , = 5 nM) for a terminal oxidase (D'mello et al., 1996). Keyword...

JapanEscherichia coli flavohaemoglobin (Hmp) reduced purified mitochondria1 cytochrome c aerobically in a reaction that was not substantially inhibited by superoxide dismutase, demonstrating that superoxide anion, the product of 0, reduction by Hmp, did not contribute markedly to cytochrome c reduction. Cytochrome c was reduced by Hmp even in the presence of 0 5 mM CO, when the haem B was locked in the ferrous, low-spin state, demonstrating that electron transfer to cytochrome c from NADH was via FAD, not haem. Hmp also reduced the ferrisiderophore complex Fe(lll)-hydroxamate K from Rhizobium leguminosanrm bv. wiciae anaerobically in a CO-insensitive manner, but at low rates and with low affinity for this substrate. The NADH+ytochrome c oxidoreductase activity of Hmp was slightly sensitive to the binding and reduction of 0, at the haem. The V-of cytochrome c reduction fell from 7.1 s-l in the presence of 0 5 mM CO to 5 0 s'l in the presence of 100 pM O,, with no significant change in Km for cytochrome c (68 to 7.3 pM, respectively). 0, at near-micromolar concentrations diminished cytochrome c reduction to a similar extent as did 100 pM 0 , . Thus, Hmp acts as a reductase of broad specificity, apparently without involvement of electron transfer via the globinlike haem. These data are consistent with the hypothesis that Hmp could act as an intracellular sensor of 0, since, in the absence of O, , electron flux from FAD to other electron acceptors increases. However, the nature of such acceptors in wivo is not known and alternative models for 0, sensing are also considered.