[Interaction of Escherichia coli cytochrome bd with hydrogen peroxide].

The absorption spectrum of the cytochrome bd complex from Escherichia coli in the "as isolated" state is characterized by an intense band at approximately 648 nm belonging to reduced heme d oxycomplex (d2+-O2). This band is often accompanied by a small shoulder around 680 nm. Treatment of the oxycomplex with hydrogen peroxide results in the loss of the 648 nm band and increased absorbance at 680 nm. The peak at 680 nm also appears in the difference absorption spectrum after addition of hydrogen peroxide to the oxidized form of the enzyme and can be attributed to formation of a peroxy or an oxoferryl complex of heme d. The increase in extinction at 680 nm is accompanied by a small red shift of the Soret band; the corresponding difference spectrum with lambda min = 405-410 nm and lambda max = 430-440 nm is of a magnitude similar to the changes in the visible region (delta A440-410 approximately equals 10 mM-1.cm-1). This circumstance favours H2O2 interaction with heme d rather than b595. The lineshape of the H2O2-induced spectral changes does not vary throughout the hydrogen peroxide concentration range studied (5 microM-5 mM). The H2O2 concentration dependence on the 680 nm peak magnitude follows a saturation curve with apparent Kd of 30-40 microM. The product of cytochrome bd interaction with H2O2 reacts with cyanide approximately tenfold slower than the free oxidized enzyme. Addition of excess catalase to the hydrogen peroxide-treated cytochrome bd complex fully reverses the H2O2-induced spectral changes. However, the rate of disappearance of these changes (keff approximately equals 10(-3) s-1) is ca. 10-fold slower than expected for the dissociation rate constant, koff, for the peroxy adduct, assuming reversible H2O2 binding with Kd approximately equal to 30-40 microM and kon > 500 M-1.s-1. This may point to H2O2 interaction with cytochrome bd, being essentially irreversible. The initial addition of H2O2 to heme d is likely to be followed by cleavage of the O--O bond, giving rise to the oxoferryl state (Fe4+ = O) of heme d which disappears upon removal of H2O2 by catalase due to reduction by endogenous electron sources.