A mutation in subunit I of cytochrome oxidase from Rhodobacter sphaeroides results in an increase in steady-state activity but completely eliminates proton pumping.

The heme-copper oxidases convert the free energy liberated in the reduction of O(2) to water into a transmembrane proton electrochemical potential (protonmotive force). One of the essential structural elements of the enzyme is the D-channel, which is thought to be the input pathway, both for protons which go to form H(2)O ("chemical protons") and for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel contains a chain of water molecules extending about 25 A from an aspartic acid (D132 in the Rhodobacter sphaeroides oxidase) near the cytoplasmic ("inside") enzyme surface to a glutamic acid (E286) in the protein interior. Mutations in which either of these acidic residues is replaced by their corresponding amides (D132N or E286Q) result in severe inhibition of enzyme activity. In the current work, an asparagine located in the D-channel has been replaced by the corresponding acid (N139 to D; N98 in bovine enzyme) with dramatic consequences. The N139D mutation not only completely eliminates proton pumping but, at the same time, confers a substantial increase (150-300%) in the steady-state cytochrome oxidase activity. The N139D mutant of the R. sphaeroides oxidase was further characterized by examining the rates of individual steps in the catalytic cycle. Under anaerobic conditions, the rate of reduction of heme a(3) in the fully oxidized enzyme, prior to the reaction with O(2), is identical to that of the wild-type oxidase and is not accelerated. However, the rate of reaction of the fully reduced enzyme with O(2) is accelerated by the N139D mutation, as shown by a more rapid F --> O transition. Whereas the rates of formation and decay of the oxygenated intermediates are altered, the nature of the oxygenated intermediates is not perturbed by the N139D mutation.