A single amino acid residue controls ROS production in the respiratory Complex I from Escherichia coli.

Reactive oxygen species (ROS) production by respiratory Complex I from Escherichia coli was studied in bacterial membrane fragments and in the isolated and purified enzyme, either solubilized or incorporated in proteoliposomes. We found that the replacement of a single amino acid residue in close proximity to the nicotinamide adenine dinucleotide (NADH)-binding catalytic site (E95 in the NuoF subunit) dramatically increases the reactivity of Complex I towards dioxygen (O2 ). In the E95Q variant short-chain ubiquinones exhibit strong artificial one-electron reduction at the catalytic site, also leading to a stronger increase in ROS production. Two mechanisms can contribute to the observed kinetic effects: (a) a change in the reactivity of flavin mononucleotide (FMN) towards dioxygen at the catalytic site, and (b) a change in the population of the ROS-generating state. We propose the existence of two (closed and open) states of the NAD(+) -bound enzyme as one feature of the substrate-binding site of Complex I. The analysis of the kinetic model of ROS production allowed us to propose that the population of Complex I with reduced FMN is always low in the wild-type enzyme even at low ambient redox potentials, minimizing the rate of reaction with O2 in contrast to E95Q variant.

Structure of Plasmodium falciparum TRAP (thrombospondin-related anonymous protein) A domain highlights distinct features in apicomplexan von Willebrand factor A homologues.

TRAP (thrombospondin-related anonymous protein), localized in the micronemes and on the surface of sporozoites of the notorious malaria parasite Plasmodium, is a key molecule upon infection of mammalian host hepatocytes and invasion of mosquito salivary glands. TRAP contains two adhesive domains responsible for host cell recognition and invasion, and is known to be essential for infectivity. In the present paper, we report high-resolution crystal structures of the A domain of Plasmodium falciparum TRAP with and without bound Mg2+. The structure reveals a vWA (von Willebrand factor A)-like fold and a functional MIDAS (metal-ion-dependent adhesion site), as well as a potential heparan sulfate-binding site. Site-directed mutagenesis and cell-attachment assays were used to investigate the functional roles of the surface epitopes discovered. The reported structures are the first determined for a complete vWA domain of parasitic origin, highlighting unique features among homologous domains from other proteins characterized hitherto. Some of these are conserved among Plasmodiae exclusively, whereas others may be common to apicomplexan organisms in general.

Role of Ca(2+) in structure and function of Complex I from Escherichia coli.

The dependence of E. coli Complex I activity on cation chelators such as EDTA, EGTA, NTA and o-phenanthroline was studied in bacterial membranes, purified solubilized enzyme and Complex I reconstituted into liposomes. Purified Complex I was strongly inhibited by EDTA with an I(50) of approximately 2.5µM. The effect of Mg(2+) and Ca(2+) on EGTA inhibition of purified Complex I activity indicated that Ca(2+) is tightly bound to the enzyme and essential for the activity. Low sensitivity to o-phenanthroline argues against the occupation of this cation binding site by Fe(2+) or Zn(2+). The sensitivity of Complex I to EDTA/EGTA strongly depends on the presence of monovalent cations in the medium, and on whether the complex is native, membrane-bound, or purified. The data is discussed in terms of a possible loss either of an additional 14th, subunit of E. coli Complex I, analogous to Nqo15 in the T. thermophilus enzyme, or another component of the native membrane that affects the affinity and/or accessibility of the Ca(2+) binding site.

Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis.

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.

Ca2+ stabilization of respiratory complex I from Escherichia coli.

Stability of the membrane-bound and purified H+-translocating NADH:ubiquinone oxidoreductase, Complex I, was studied. The loss of the enzyme activity is strongly increased by alkaline pH and dilution of the sample. Complex I inactivation is prevented specifically by a low concentration of Ca2+ and/or an intracellular stabilization factor (ISF). The action of both, Ca2+ and ISF, on Complex I stability is interdependent. The data are discussed in terms of a release of structural Ca2+ as a reason for Complex I decay and an effect of ISF on the affinity and/or accessibility of Ca2+-binding site.