Defective energy coupling in delta-subunit mutants of Escherichia coli F1F0-ATP synthase.

A.L Hazard,A.E Senior

doi:10.1016/s0021-9258(17)42368-4

A.L Hazard, A.E Senior

Open Access

https://doi.org/10.1016/s0021-9258(17)42368-4

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Abstract

Membrane vesicles from 13 strains carrying mutations in the C-terminal region of the delta-subunit of Escherichia coli F1F0-ATP synthase were characterized in respect to ATPase activity, ATP-driven proton-pumping, dicyclohexylcarbodiimide sensitivity of ATPase, and oxidative phosphorylation. The salient finding was that energy-coupling between F1 and F0 sectors of the enzyme is impaired by several of the mutations. The delta G150N mutant appeared completely uncoupled in vitro. The data emphasize the role of the C-terminal region of delta-subunit in integration of the proton conduction machinery in F0 with the three F1 catalytic sites. It is suggested that the C-terminal region of delta-subunit, speculatively located in the central region of the alpha 3 beta 3 hexagon, acts functionally at the interface between the helical domain of the stalk and the F1 subunits to relay conformational signals which alter the affinities of the catalytic sites for substrates and products.

Highlights

The previous paper (Hazard and Senior, 1994) describtehse tions in the C-terminal regioonf the &subunitof Esch- generation of mutations in the C-terminal region of Ssubunit erichia coli FIFo-ATPsynthasewerecharacterizedin by random, cassette, and site-directed mutagenesis
Mutants exemplifying a range of &subunit in integration of the proton conduction ma- growth characteristics were chosen for analysis, includfiinvge chinery inFo with the threeF1 catalytic sites.It is sug- mutants which showed zero or very low growth by oxidative gested that the C-terminal regioonf &subunit, specula- phosphorylation (6G150T, sG150P, sG150D, 6G150N, and tively located in the central regioonf the aspshexagon, SL99P/A149D), three mutants with intermediate growth characts functionally at the interface between the helical acteristics (6L99P, SGlSOA, a n d SG150CN164L), and five mudomain of the stalk and theF1 subunits to relay confor-tants with relatively strong growth characteristics (SG150S, mational signals which alter the affinities of the cata- 6A149P, SA149S, 6A149T, and 6A149D)
Sectors, theF1 sector which catalyzesthe hydrolysis or synthe- Growth of E. coli Cells;Preparation of Membrane Vesicles-Bacterial sis of ATP, a n d the Fosector which conducts protons acrostshe strains and growth conditions were as described in the accompanying membrane

Summary

RESULTS

Driven proton pumping in KSCN-extracted and F1-reconstituted membranes, and low amounts of subunit b in the mem-. F1may be firmly spectively, in Table I, column 2 These data showed that the attached toFo,but ATPase activity maybe partly or completely observed impairment of energy coupling was independent of uncoupled from proton movement, so that DCCD binding t o Fo the level of expression of the enzyme and was caused by the does not fully inhibit ATPase activity. For the purposes of this study, howminor (5-15%) and inconsistent reversal of the inhibition of ever, the lack of a sizeable and consistent reversal of inhibition ATPase was seen It was confirmed, using the acridine orange by NADH or succinate in wild-type membranes prevented us fluorescence quench test, that a transmembrane proton gradi- from applying this assay to mutants.

DISCUSSION

Energy coupling properties

The key feature of the mechanism for ATP synthesis is thAatTP