ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter.

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Impact of the iron-sulfur cluster proximal to the active site on the catalytic function of an O2-tolerant NAD(+)-reducing [NiFe]-hydrogenase.

The soluble NAD(+)-reducing hydrogenase (SH) from Ralstonia eutropha H16 belongs to the O2-tolerant subtype of pyridine nucleotide-dependent [NiFe]-hydrogenases. To identify molecular determinants for the O2 tolerance of this enzyme, we introduced single amino acids exchanges in the SH small hydrogenase subunit. The resulting mutant strains and proteins were investigated with respect to their physiological, biochemical, and spectroscopic properties. Replacement of the four invariant conserved cysteine residues, Cys41, Cys44, Cys113, and Cys179, led to unstable protein, strongly supporting their involvement in the coordination of the iron-sulfur cluster proximal to the catalytic [NiFe] center. The Cys41Ser exchange, however, resulted in an SH variant that displayed up to 10% of wild-type activity, suggesting that the coordinating role of Cys41 might be partly substituted by the nearby Cys39 residue, which is present only in O2-tolerant pyridine nucleotide-dependent [NiFe]-hydrogenases. Indeed, SH variants carrying glycine, alanine, or serine in place of Cys39 showed increased O2 sensitivity compared to that of the wild-type enzyme. Substitution of further amino acids typical for O2-tolerant SH representatives did not greatly affect the H2-oxidizing activity in the presence of O2. Remarkably, all mutant enzymes investigated by electron paramagnetic resonance spectroscopy did not reveal significant spectral changes in relation to wild-type SH, showing that the proximal iron-sulfur cluster does not contribute to the wild-type spectrum. Interestingly, exchange of Trp42 by serine resulted in a completely redox-inactive [NiFe] site, as revealed by infrared spectroscopy and H2/D(+) exchange experiments. The possible role of this residue in electron and/or proton transfer is discussed.