Proton exit from the heme-copper oxidase of Escherichia coli

Pathways of proton entry have been identified in the proton-translocating h eme-copper oxidases, but the proton exit pathway is unknown. Here we report experiments with cytochrome bos in Escherichia coli cells that may identif y the beginning of the exit pathway. Systematic mutations of arginines 438 and 439 (R481 and R482 in the E. coli enzyme), numbering as in cytochrome a a(3) from bovine heart mitochondria, which interact with the ring D propion ates of the two heme groups, reveal that the D propionate of the oxygen-bin ding heme is involved in proton pumping; its anionic form must be stabilize d in order for proton translocation to occur. This may locate the beginning of the pathway by which pumped protons exit from the enzyme structure.