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Title: Studies on the cytochrome oxidase of Pseudomonas stutzeri
Author: Hunter, Dominic J. B.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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The presence of an o-type cytochrome oxidase in the membranes of the denitrifying soil bacterium Pseudomonas stutzeri 224 was suggested by difference spectroscopy and by carbon monoxide binding spectra. A small amount of a d-type cytochrome was also detected. The sensitivities of the oxidase activities of the membranes of P.stutzeri to cyanide leads to the conclusion that cytochrome d is associated with NADH oxidase activity and cytochrome o is associated with ascorbate-TMPD oxidase activity. Cytochrome c4 has been implicated in the o-type oxidase of Azotobacter vinelandii (Jurtshuk et al., 1981). The role of this cytochrome in the P.stutzeri o-type oxidase was investigated by selective removal of the cytochrome c4 from P.stutzeri membranes. Sodium iodide, at concentrations above 1.5M, and propan-2-ol, at concentrations above 20% (v/v) were found to remove cytochrome c4 from P.stutzeri membranes selectively with respect to other c-type cytochromes, as judged by haem-stained SDS-PAGE gels. Cytochrome c4 could also be removed from octyl glucoside-solubilised membranes after frozen storage by chromatography on Sephadex G-75. Removal did not occur when freshly solubilised membranes were used. Ascorbate-TMPD and cytochromes c oxidase activities of the membranes were retained, despite the removal of cytochrome c4. Duroquinol oxidase activity was partially lost, whilst NADH, succinate and lactate oxidase activities were completely lost. Purified cytochrome c4, or that removed by iodide treatment, could be reconstituted with cytochrome c4-depleted membranes with no effect on the oxidase activities of the membrane. Return of purified cytochrome c4 was shown to be by a specific mechanism, rather than being due to non-specific co-precipitation with the membranes. It was therefore concluded that cytochrome c4 is not involved in the ascorbate-TMPD or cytochromes c oxidase activities. The losses of duroquinol, NADH, succinate and lactate oxidase activities are probably due to damage to other components of the electron transport chain, since the treatments used to remove cytochrome c4 remove other non-haem proteins in addition. This conclusion implies that cytochrome c4 is not involved in the o-type cytochrome oxidase of P.stutzeri. A similar conclusion can be drawn for A.vinelandii (Hunter et al., 1989). Iodide treatment of cytochrome c4 caused its partial denaturation. The midpoint potentials of the cytochrome c4 in sodium iodide (+5 and -80mV) were considerably lower than the native values (+310 and +195mV), indicating a greater exposure of the haems to solvent. Additionally, the near-infrared band of the ferricytochrome, indicative of methionyl-haem coordination, was lost on iodide treatment. Removal of the iodide allowed 60&37 of the cytochrome c4 to regain its native properties. Purification of the o-type oxidase of P.stutzeri 224 was undertaken. The ascorbate-TMPD oxidase activity was solubilised intact by octyl glucoside, dodecyl maltoside and Triton X-100, but not by CHAPS, MEGA-9 or deoxycholate. When solubilised by dodecyl maltoside and Triton X-100, the oxidase proved refractory to purification. The following methods were also of no use in the purification:affinity chromatography on yeast cytochrome c-sepharose; hydrophobic chromatography on octyl Sepharose; chromatography on hydroxyapatite; ion exchange chromatography of the crude detergent extract; molecular exclusion chromatography of the crude detergent extract and molecular exclusion chromatography on columns equilibrated with octyl glucoside. The ascorbate-TMPD oxidase was partially purified by ammonium sulphate fractionation of an octyl glucoside extract, the oxidase activity precipitating as a red oil at 70% saturation. This step removed most of the cytochromes c, along with many of the proteins of the extract. Further purification was achieved by molecular exclusion chromatography on Fractogel HW-55 in Triton X-100, which was capable of separating much of the remainder of the cytochromes c from the oxidase. This step was followed by ion exchange chromatography on DE-cellulose. The final product contained cytochromes b and c and possessed cyanide inhibitable ascorbate-TMPD oxidase activity. The polypeptide composition of this material was too complex to permit identification of the subunits of the oxidase. The removal of most of the cytochrome c4 from the oxidase activity during this preparation supports the conclusion that cytochrome c4 is not involved in the o-type oxidase in P.stutzeri. In the light of this, the possible function of cytochrome c4 as a link between the o-type oxidase and its reductase is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available