Electron-bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate potential electron donor are split so that one is sent along a high potential pathway to a high potential acceptor and the other is sent along a low potential pathway to a low potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognised, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn2+ site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron-bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.

Ultrafast two-dimensional infrared (2D-IR) spectroscopy of Escherichia coli Hyd-1 (EcHyd-1) reveals the structural and dynamic influence of the protein scaffold on the Fe(CO)(CN)2unit of the active site. Measurements on...

Verteporfin, a free base benzoporphyrin derivative monoacid ring A, is a photosensitizing drug for photodynamic therapy (PDT) used in the treatment of the wet form of macular degeneration and activated by red light of 689 nm. Here, we present the first direct study of its photofragmentation channels in the gas phase, conducted using a laser interfaced mass spectrometer across a broad photoexcitation range from 250 to 790 nm. The photofragmentation channels are compared with the collision-induced dissociation (CID) products revealing similar dissociation pathways characterized by the loss of the carboxyl and ester groups. Complementary solution-phase photolysis experiments indicate that photobleaching occurs in verteporfin in acetonitrile; a notable conclusion, as photoinduced activity in Verteporfin was not thought to occur in homogenous solvent conditions. These results provide unique new information on the thermal break-down products and photoproducts of this light-triggered drug.

Electron-bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate potential electron donor are split so that one is sent along a high potential pathway to a high potential acceptor and the other is sent along a low potential pathway to a low potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognised, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers electrically connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a “closed bridge” and an “open bridge” conformation, where a Zn2+ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron-bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD+ reduction/NADH oxidation site.