The F ₁ F _O and F ₁ ‐ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome‐reconstituted holoenzyme preserves its tight coupling between F ₁ and F _O sectors, as evidenced by its high sensitivity to the F _O inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N‐terminal sequencing of the band profile in SDS‐PAGE of the F ₁ and F ₁ F _O preparations showed a novel 11‐kDa protein in addition to the 5 canonical α, β, γ, δ, and ε subunits present in all known F ₁ ‐ATPase complexes. BN‐PAGE followed by 2D‐SDS‐PAGE confirmed the presence of this 11‐kDa protein bound to the native F ₁ F _O ‐ATP synthase of P. denitrificans , as it was observed after being isolated. The recombinant 11 kDa and ε subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F ₁ F _O and soluble F ₁ ‐ATPase complexes. The 11‐kDa protein, but not the ε subunit, inhibited the F ₁ F _O and F ₁ ‐ATPase activities of P. denitrificans . The 11‐kDa protein was also found in Rhodobacter sphaeroides associated to its native F ₁ F _O ‐ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11‐kDa protein on the F ₁ F _O ‐ATPase nanomotor of P. denitrificans and closely related α‐proteobacteria.—Morales‐Ríos, E., de la Rosa‐Morales, F., Mendoza‐Hernández, G., Rodríguez‐Zavala, J. S., Celis, H., Zarco‐Zavala, M., García‐Trejo, J. J. A novel 11‐kDa inhibitory subunit in the F ₁ F _O ATP synthase of Paracoccus denitrificans and related α‐proteobacteria. FASEB J . 24, 599–608 (2010). www.fasebj.org

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c(10) ring and the other stator subunits at the F(0)-F(0) dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356-12358). In the ATP synthase dimer, the two peripheral stalks are located near the F(1)-F(1) interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.

Significance: F-ATP synthase is a fundamental enzyme supplying adenosine triphosphate (ATP), spreading across all kingdoms of life. Despite remarkable conservation of its basic structure and function, biophysical studies have revealed discrete differences in the rotary mechanisms of bacterial and eukaryotic F ₁ -ATPases (the catalytic portions of the enzymes). Here, we analyzed the rotational dynamics of Paracoccus denitrificans F ₁ (PdF ₁ ), a bacterial F ₁ -ATPase that exhibits high homology with the core functional subunits of its mitochondrial counterpart. Notably, PdF ₁ possesses a simplified chemomechanical scheme different from that of all other F ₁ -ATPases. Our results reveal an unexpected diversity in the chemomechanical coupling cycle of the F ₁ -ATPase machinery and show that features such as homology or phylogenetic relationship cannot uniquely define the rotary scheme pattern.

The ζ subunit is a novel natural inhibitor of the α‐proteobacterial F ₁ F _O ‐ATPase described originally in Paracoccus denitrificans . To characterize the mechanism by which this subunit inhibits the F ₁ F _O nanomotor, the ζ subunit of Paracoccus denitrificans (Pd‐ζ was analyzed by the combination of kinetic, biochemical, bioinformatic, proteomic, and structural approaches. The ζ subunit causes full inhibition of the sulfite‐activated PdF ₁ ‐ATPase with an apparent IC ₅₀ of 270 nM by a mechanism independent of the 8 subunit. The inhibitory region of the ζ subunit resides in the first 14 N‐terminal residues of the protein, which protrude from the 4‐α‐helix bundle structure of the isolated ζ subunit, as resolved by NMR. Cross‐linking experiments show that the ζ subunit interacts with rotor (γ)and stator (α, β) subunits of the F ₁ ‐ATPase, indicating that the ζ subunit hinders rotation of the central stalk. In addition, a putatively regulatory nucleotide‐binding site was found in the ζ subunit by isothermal titration calorimetry. Together, the data show that the ζ subunit controls the rotation of F ₁ F _O ‐ATPase by a mechanism reminiscent of, but different from, those described for mitochondrial IF ₁ and bacterial ε subunits where the 4‐α‐helix bundle of ζ seems to work as an anchoring domain that orients the N‐terminal inhibitory domain to hinder rotation of the central stalk.—Zarco‐Zavala, M., Morales‐Ríos, E., Mendoza‐Hernández, G., Ramírez‐Silva, L., Pérez‐Hernández, G., García‐Trejo, J. J. The ζ subunit of the F ₁ F _O ‐ATP synthase of α‐proteobacteria controls rotation of the nanomotor with a different structure. FASEB J . 28, 2146–2157 (2014). www.fasebj.org

The biological roles of the three natural F1FO-ATPase inhibitors, ε, ζ, and IF1, on cell physiology remain controversial. The ζ subunit is a useful model for deletion studies since it mimics mitochondrial IF1, but in the F1FO-ATPase of Paracoccus denitrificans (PdF1FO), it is a monogenic and supernumerary subunit. Here, we constructed a P. denitrificans 1222 derivative (PdΔζ) with a deleted ζ gene to determine its role in cell growth and bioenergetics. The results show that the lack of ζ in vivo strongly restricts respiratory P. denitrificans growth, and this is restored by complementation in trans with an exogenous ζ gene. Removal of ζ increased the coupled PdF1FO-ATPase activity without affecting the PdF1FO-ATP synthase turnover, and the latter was not affected at all by ζ reconstitution in vitro. Therefore, ζ works as a unidirectional pawl-ratchet inhibitor of the PdF1FO-ATPase nanomotor favoring the ATP synthase turnover to improve respiratory cell growth and bioenergetics.

Lamivudine, also widely known as 3TC belongs to a family of nucleotide/nucleoside analogues of cytidine or cytosine that inhibits the Reverse Transcriptase (RT) of retroviruses such as HIV. Lamivudine is currently indicated in combination with other antiretroviral agents for the treatment of HIV-1 infection or for chronic Hepatitis B (HBV) virus infection associated with evidence of hepatitis B viral replication and active liver inflammation. HBV reactivation in patients with HBV infections who receive anticancer chemotherapy can be a life-threatening complication during and after the completion of chemotherapy. Lamivudine is used, as well as other antiretrovirals, to prevent the reactivation of the Hepatitis B virus during and after chemotherapy. In addition, Lamivudine has been shown to sensitize cancer cells to chemotherapy. Lamivudine and other similar analogues also have direct positive effects in the prevention of cancer in hepatitis B or HIV positive patients, independently of chemotherapy or radiotherapy. Recently, it has been proposed that Lamivudine might be also repurposed against SARS-CoV-2 in the context of the COVID-19 pandemic. In this review we first examine recent reports on the re-usage of Lamivudine or 3TC against the SARS-CoV-2, and we present docking evidence carried out in silico suggesting that Lamivudine may bind and possibly work as an inhibitor of the SARS-CoV-2 RdRp RNA polymerase. We also evaluate and propose assessment of repurposing Lamivudine as anti-SARS-CoV-2 and anti-COVID-19 antiviral. Secondly, we summarize the published literature on the use of Lamivudine or (3TC) before or during chemotherapy to prevent reactivation of HBV, and examine reports of enhanced effectiveness of radiotherapy in combination with Lamivudine treatment against the cancerous cells or tissues. We show that the anti-cancer properties of Lamivudine are well established, whereas its putative anti-COVID effect is under investigation. The side effects of lamivudine and the appearance of resistance to 3TC are also discussed.