Probing interactions of the Escherichia coli FoFI ATP synthase β and γ subunits with disulphide cross-links

Abstract

As indicated above, there was a time in the pioneering days of the 1960s that many coupling factors, e.g. F2, F3, F4 and F5, which are not known today, were being considered as serious candidates for key roles in oxidative phosphorylation. Beechey was not convinced as to the validity of many of these claims and argued [13-151 that many of the factors were distinguished only by a differential enrichment of one particular component, e.g. the OSCP. He did much to clarify what was, and what was not, a discrete coupling factor.Brian Beechey's work in the ATPase field (and by no means all of it can be summarized here: see for example Lowe and Beechey [16] for a later contribution), and others in which he has worked such as investigating the claim that iodohistidine was an intermediate in oxidative phosphorylation [ 171 , is characterized by its careful and thorough execution. This has meant that it has stood the test of time and has, therefore, contributed very significantly indeed to what we know today about the ATP synthase.

A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis

“…In the case of ␤E381C, the activity level for the DTT-treated enzyme was higher than the initial rate because of complete reduction of the small fraction of crosslinked enzyme present prior to addition of CuCl 2 . There was no effect on wild-type steady state membrane ATPase activity by CuCl 2 , DTNB, or DTT, as shown previously (27).…”

“…To assay the rotation steps, we decided to use a rotor-stator, ␥ subunit to ␤ subunit, disulfide bond, with the premise that a cross-link would block rotation and therefore stop the partial reactions involving rotation. Such disulfides have been previously observed between the native Cys at position 87 in the Escherichia coli ␥ subunit and Cys substitutions in the conserved 380 ␤DELSEED 386 motif, either at ␤Asp-380 (27) or ␤Glu-381 (28) (Fig. 1).…”

“…f ND means experiment was not repeated here. Duncan et al (27) have already shown there is no effect of either DTNB or DTT on wild-type activity. g Activity was fully inhibitable by 1 mM sodium azide, indicating the extent of uncross-linked enzyme.…”

“…Enzymes-It was previously shown that a disulfide bond can be induced between the native ␥ subunit Cys-87 and a Cys substitution in place of either ␤ subunit Asp-380 (27,40) or Glu-381 (28) (Fig. 1).…”

Conformational Changes in the Escherichia coli ATP Synthase (ECF1F0) Monitored by Nucleotide-dependent Differences in the Reactivity of Cys-87 of the γ Subunit in the Mutant βGlu-381 → Ala

“…Recently, Duncan et al (1995aDuncan et al ( , 1995b) used a mutant ␤D380C/␥C87S to distinguish which of the two Cys in the ␥ subunit (Cys-87 or Cys-112) was involved in disulfide bond cross-linking between ␥ and the ␤ DELSEED region. Following the same approach, we constructed the mutant ␤E381C/␥C87S/ ⑀S108C to identify which of the intrinsic Cys in ␥ was being reacted by maleimides.…”