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Redox poise and oxygenation of cytochrome bd in the diazotroph Azotobacter vinelandii assessed in vivo using diode-array reflectance spectrophotometry

James B. Callis^1,

Nitrogen Fixation Laboratory, John Innes Centre, Norwich NR4 7UH, UK
Division of Life Sciences, King's College London, Campden Hill Road, London W8 7AH, UK
Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, UK

ABSTRACT

A ferrous oxygenated form of cytochrome d is characteristic of all cytochrome bd-type oxidases so far examined, but its participation in enzyme turnover is unclear. It is relatively stable, occurs in aerated cell suspensions and predominates during enzyme preparation. In this study, diode-array reflectance spectrophotometry was used to assess the redox poise and oxygenation of cytochrome bd in vivo, in the aerobic diazotroph Azotobacter vinelandii. Mutants either lacking or overproducing the cytochrome bd oxidase were used to confirm the reliability of the optical configuration. Changes in absorbance attributed to cytochromes b, c and d were followed as the O₂ supply was altered either in suspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d, which differed in absorbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 630 nm and (3) the reduced form that absorbs at 630 nm. The transition between states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b₅₉₅ and b₅₆₀. The dissolved O₂ concentration at which this transition occurred coincided approximately with the apparent O₂ affinity for the oxidase in vivo (approx. 5 µM). During steady-state growth, the cytochromes were partially reduced and the oxygenated form of cytochrome d was undetected. These in situ measurements support the view that an oxygenated form of cytochrome d (absorbing at 650 nm) in the one-electron-reduced cytochrome bd-type oxidase does not take part in enzyme turnover.

^*Author for correspondence: Susan Hill. Tel: +44 1273 474017. Fax: +44 1603 454970. e-mail: shill@pavilion.co.uk

Present address: Department of Chemistry, University of Washington, Seattle, WA 98195, USA.