Abolishment of proton pumping and accumulation in the E1P conformational state of a plant plasma membrane H+-ATPase by substitution of a conserved aspartyl residue in transmembrane segment 6

The plasma membrane H+-ATPase AHA2 ofArabidopsis thaliana, which belongs to the P-type ATPase superfamily of cation-transporting ATPases, pumps protons out of the cell, To investigate the mechanism of ion transport by P-type ATPases we have mutagenized Asp(684), a residue in transmembrane segment M6 of AHA2 that is conserved in Ca2+-, Na+/K+-, H+/K-, and H+-ATPases and which coordinates Ca2+ ions in the SERCA1 Ca2+-ATPase. We describe the expression, purification, and biochemical analysis of the Asp(684) -->Asn mutant, and provide evidence that Asp(684) in the plasma membrane H+-ATPase is required for any coupling between ATP hydrolysis, enzyme conformational changes, and H+-transport. Proton pumping by the reconstituted mutant enzyme was completely abolished, whereas ATP was still hydrolyzed, The mutant was insensitive to the inhibitor vanadate, which preferentially binds to P-type ATPases in the E-2 conformation. During catalysis the Asp684, Asn enzyme accumulated a phosphorylated intermediate whose stability was sensitive to addition of ADP. We conclude that the mutant enzyme is locked in the E-1 conformation and is unable to proceed through the E1P-E2P transition.