The conserved Glu540 in subunit I of Escherichia coli cytochrome bo (a H+ pump) is replaced by Asp544 in the Vitreoscilla enzyme (a Na+ pump). Site-directed mutagenesis of the Vitreoscilla cytochrome bo operon changed this Asp to Glu, and both wild type and mutant cyo's were transformed into E. coli strain GV100 which lacks cytochrome bo. Compared to the wild type transformant the Asp544Glu transformant had decreased ability to pump Na+ as well as decreased stimulation in respiratory activity in the presence of Na+. Preliminary experiments indicated that this mutant also had increased ability to pump protons, suggesting that this single change may provide cation pumping specificity in this group of enzymes. (c) 2006 Published by Elsevier Inc.