Abstract
Homology models of plasma membrane H(+)-ATPase (Bukrinsky, J. T., Buch-Pedersen, M. J., Larsen, S., and Palmgren, M. G. (2001) FEBS Lett. 494, 6-10) has pointed to residues in transmembrane segment M4 as being important for proton translocation by P-type proton pumps. To test this model, alanine-scanning mutagenesis was carried out through 12 residues in the M4 of the plant plasma membrane H(+)-ATPase AHA2. An I282A mutation showed apparent reduced H(+) affinity, and this residue was subsequently substituted with all other naturally occurring amino acids by saturation mutagenesis. The ability of mutant enzymes to substitute for the yeast proton pump PMA1 was found to correlate with the size of the side chain rather than its chemical nature. Thus, smaller side chains (Gly, Ala, and Ser) at this position resulted in lower H(+) affinity and lowered levels of H(+) transport in vivo, whereas substitution with side chains of similar and larger size resulted in only minor effects. Substitutions of Ile-282 had only minor effects on ATP affinity and sensitivity toward vanadate, ruling out an indirect effect through changes in the enzyme conformational equilibrium. These results are consistent with a model in which the backbone carbonyl oxygen of Ile-282 contributes directly to proton translocation.
Highlights
Plasma membrane proton ATPases are vital proteins for plant and fungal life
The transported proton is likely to get occluded during catalysis in the form of a hydronium ion in a specific high affinity hydronium ion coordination center (5)
No hydronium coordination center in a protein has been described to date, but high affinity hydronium ion coordination centers have been demonstrated in specific crown ether structures (11, 12) in which the hydronium ion is coordinated by the use of hydrogen bonds
Summary
Plasma membrane proton ATPases are vital proteins for plant and fungal life. These pumps are responsible for ATPfueled ejection of protons out of the cell and establish the essential proton and electrical gradient across plant and fungal plasma membranes. The transport mechanism of P-type ATPases has been described as an alternating access mechanism in which one or more cation coordination centers change affinity for the transported ion(s) (1) In these coordination centers, the transported cations get tightly bound within the membrane sector of the protein during catalysis in a process termed occlusion. The transported cations get tightly bound within the membrane sector of the protein during catalysis in a process termed occlusion The nature of such a P-type ATPase coordination center is most clearly seen in the high resolution structure of the sarcoplasmic reticulum Ca2ϩ-ATPase in which oxygen atoms from both the side chains and the main chain carbonyls provide liganding groups for the coordination of two calcium ions (10). No hydronium coordination center in a protein has been described to date, but high affinity hydronium ion coordination centers have been demonstrated in specific crown ether structures (11, 12) in which the hydronium ion is coordinated by the use of hydrogen bonds
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