The Reaction Of Cytochrome aa3-600 With Radical Trapping Agents

Abstract

Cytochrome aa3-600 or menaquinol oxidase from Bacillus subtilis is a member of the heme-copper oxidase family that includes mitochondrial cytochrome c oxidase. A distinguishing feature of cytochrome aa3-600 is that it does not oxidize cytochrome c and does not contain a CuA center, but instead uses menaquinol as reducing substrate to convert O2 to water. A radical signal is observed when cytochrome aa3-600 is frozen during the course of steady-state catalysis. The nature of this radical is not fully characterized and we aim to understand it further by using the radical traps 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 2-methyl-2-nitrosopropane (MNP) and N-tert-butyl-phenylnitrone (BPN). TEMPO appears to inhibit menaquinol oxidase's steady state activity, whereas MNP and BPN are without effect. Heme-copper oxidases form a series of intermediates when exposed to H2O2 that are related to the intermediates formed in the much faster reaction with oxygen. Addition of H2O2 to oxidized cytochrome aa3-600 leads to formation of the “P-state” (606 nm), which is followed by progression to the “F-state” (580 nm). The progress of this reaction is halted at the P-state when performed in the presence of TEMPO (50 μM- 5mM). In addition if TEMPO is added at the end of the H2O2 reaction the F-state is converted back to the P-state. We propose that the inhibition of cytochrome aa3-600 by TEMPO is mediated by its ability to trap the P-state of the enzyme and slow its progress through the catalytic cycle.