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Adenosine triphosphate (ATP), the cellular energy currency, is essential for life. The ability to provide a constant supply of ATP is therefore crucial for the construction of artificial cells in synthetic biology. Here, we describe the bottom-up assembly and characterization of a minimal respiratory system that uses NADH as a fuel to produce ATP from ADP and inorganic phosphate, and is thus capable of sustaining both upstream metabolic processes that rely on NAD + , and downstream energy-demanding processes that are powered by ATP hydrolysis. A detergent-mediated approach was used to co-reconstitute respiratory mitochondrial complex I and an F-type ATP synthase into nanosized liposomes. Addition of the alternative oxidase to the resulting proteoliposomes produced a minimal artificial "organelle" that reproduces the energy-converting catalytic reactions of the mitochondrial respiratory chain: NADH oxidation, ubiquinone cycling, oxygen reduction, proton pumping, and ATP synthesis. As a proof-of-principle, we demonstrate that our nanovesicles are capable of using an NAD + -linked substrate to drive cell-free protein expression. Our nanovesicles are both efficient and durable and may be applied to sustain artificial cells in future work.

We have investigated the role of redox cooperativity in defining the functional relationship among the three membrane-associated prosthetic groups of Escherichia coli nitrate reductase A: the two hemes (bD and bP) of the membrane anchor subunit (NarI) and the [3Fe-4S] cluster (FS4) of the electron-transfer subunit (NarH). Previously published analyses of potentiometric titrations have exhibited the following anomalous behaviors: (i) fits of titration data for heme bp and the [3Fe-4S] cluster exhibited two apparent components; (ii) heme bD titrated with an apparent electron stoichiometry (n) of <1.0; and (iii) the binding of quinol oxidation inhibitors shifted the reduction potentials of both hemes despite there being only a single quinol oxidation site (Q-site) in close juxtaposition with heme bD. Furthermore, both hemes appeared to be affected despite the absence of major structural shifts upon inhibitor binding, as judged by X-ray crystallography, or evidence of a second Q-site in the vicinity of heme bP. In a re-examination of the redox behavior of hemes bD and bP and FS4, we have developed a cooperative redox model of cofactor interaction. We show that anticooperative interactions provide an explanation for the anomalous behavior. We propose that the role of such anticooperative redox behavior in vivo is to facilitate transmembrane electron transfer across an energy-conserving membrane against an electrochemical potential.

The membrane subunit (NarI) of Escherichia coli nitrate reductase A (NarGHI) contains two b-type hemes, both of which are the highly anisotropic low-spin type. Heme bD is distal to NarGH and constitutes part of the quinone binding and oxidation site (Q-site) through the axially coordinating histidine-66 residue and one of the heme bD propionate groups. Bound quinone participates in hydrogen bonds with both the imidazole of His66 and the heme propionate, rendering the EPR spectrum of the heme bD sensitive to Q-site occupancy. As such, we hypothesize that the heterogeneity in the heme bD EPR signal arises from the differential occupancy of the Q-site. In agreement with this, the heterogeneity is dependent upon growth conditions but is still apparent when NarGHI is expressed in a strain lacking cardiolipin. Furthermore, this heterogeneity is sensitive to Q-site variants, NarI-G65A and NarI-K86A, and is collapsible by the binding of inhibitors. We found that the two main gz components of heme bD exhibit differences in reduction potential and pH dependence, which we posit is due to differential Q-site occupancy. Specifically, in a quinone-bound state, heme bD exhibits an Em,8 of -35 mV and a pH dependence of -40 mV pH(-1). In the quinone-free state, however, heme bD titrates with an Em,8 of +25 mV and a pH dependence of -59 mV pH(-1). We hypothesize that quinone binding modulates the electrochemical properties of heme bD as well as its EPR properties.