REDOX POISE AND OXYGENATION OF CYTOCHROME BD IN THE DIAZOTROPH AZOTOBACTER-VINELANDII ASSESSED IN-VIVO USING DIODE-ARRAY REFLECTANCE SPECTROPHOTOMETRY

A ferrous oxygenated form of cytochrome d is characteristic of all cyt ochrome bd-type oxidases so far examined, but its participation in enz yme turnover is unclear. It is relatively stable, occurs in aerated ce ll suspensions and predominates during enzyme preparation. In this stu dy, diode-array reflectance spectrophotometry was used to assess the r edox poise and oxygenation of cytochrome bd in vivo, in the aerobic di azotroph Azotobacter vinelandii, Mutants either lacking or overproduci ng the cytochrome bd oxidase were used to confirm the reliability of t he optical configuration. Changes in absorbance attributed to cytochro mes b, c and d were followed as the O-2 supply was altered either in s uspensions of harvested cells or during steady-state growth. In washed cell suspensions, three states of cytochrome d,which differed in abso rbance characteristics, were seen: (1) an oxygenated form that absorbs at 650 nm, (2) a form which has little absorbance at either 650 or 63 0 nm and (3) the reduced form that absorbs at 630 nm. The transition b etween states 2 and 3, but not 1 and 2, correlated with the changes in the redox states of cytochromes b(595) and b(560). The dissolved O-2 concentration at which this transition occurred coincided approximatel y with the apparent O-2 affinity for the oxidase in vivo (approx. 5 mu M). During steady-state growth, the cytochromes were partially reduce d and the oxygenated form of cytochrome d was undetected, These in sit u 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.