Abstract Anaerobic microorganisms of the Geobacter genus are effective electron sources for the synthesis of nanoparticles, for bioremediation of polluted water, and for the production of electricity in fuel cells. In multistep reactions, electrons are transferred via iron/heme cofactors of c-type cytochromes from the inner cell membrane to extracellular metal ions, which are bound to outer membrane cytochromes. We measured electron production and electron flux rates to 5×105 e s−1 per G. sulfurreducens. Remarkably, these rates are independent of the oxidants, and follow zero order kinetics. It turned out that the microorganisms regulate electron flux rates by increasing their Fe2+/Fe3+ ratios in the multiheme cytochromes whenever the activity of the extracellular metal oxidants is diminished. By this mechanism the respiration remains constant even when oxidizing conditions are changing. This homeostasis is a vital condition for living systems, and makes G. sulfurreducens a versatile electron source.

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (Kd ≈ 0.04−0.08 μM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (Kd ≈ 0.3 μM for GSU0582 versus 17 μM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

Lactoperoxidase (LPO) is a structurally complex and stable mammalian redox enzyme. Here we aim at evaluating the influence of ionic interactions and how these intertwine with the structural dynamics, stability and activity of LPO. In this respect, we have compared LPO guanidinium hydrochloride (GdmCl) and urea denaturation pathways and performed a detailed investigation on the effects of pH on the LPO conformational dynamics and stability. Our experimental findings using far-UV CD, Trp fluorescence emission and ESR spectroscopies clearly indicate that LPO charged-denaturation with GdmCl induced a sharp two-step process versus a three-step unfolding mechanism induced by urea. This differential effect between GdmCl and urea suggests that ionic interactions must play a rather prominent role in the stabilization of LPO. With both denaturants, the protein core was shown to retain activity up to near the respective Cm values. Moreover, a pH titration of LPO evidenced no significant conformational alterations or perturbation of heme activity within the 4 to 11 pH interval. In contrast, alterations of ionic interactions by poising LPO at pH 3, 2 and 12 resulted in a loss of secondary structure, loosening of tertiary contacts and loss of activity, which appear to be associated with the perturbation of the hydrophobic core, as evidenced by ANS binding, as well as disruption of the heme pocket demonstrated by optical and EPR spectroscopies. Overall, LPO is characterised by a high degree of peripheral structural plasticity without perturbation of the core heme moiety. The possible physiological meaning of such features is discussed.

The purple bacterium Rhodopseudomonas palustris TIE-1 expresses multiple small high-potential redox proteins during photoautotrophic growth, including two high-potential iron-sulfur proteins (HiPIPs) (PioC and Rpal_4085) and a cytochrome c2. We evaluated the role of these proteins in TIE-1 through genetic, physiological, and biochemical analyses. Deleting the gene encoding cytochrome c2 resulted in a loss of photosynthetic ability by TIE-1, indicating that this protein cannot be replaced by either HiPIP in cyclic electron flow. PioC was previously implicated in photoferrotrophy, an unusual form of photosynthesis in which reducing power is provided through ferrous iron oxidation. Using cyclic voltammetry (CV), electron paramagnetic resonance (EPR) spectroscopy, and flash-induced spectrometry, we show that PioC has a midpoint potential of 450 mV, contains all the typical features of a HiPIP, and can reduce the reaction centers of membrane suspensions in a light-dependent manner at a much lower rate than cytochrome c2. These data support the hypothesis that PioC linearly transfers electrons from iron, while cytochrome c2 is required for cyclic electron flow. Rpal_4085, despite having spectroscopic characteristics and a reduction potential similar to those of PioC, is unable to reduce the reaction center. Rpal_4085 is upregulated by the divalent metals Fe(II), Ni(II), and Co(II), suggesting that it might play a role in sensing or oxidizing metals in the periplasm. Taken together, our results suggest that these three small electron transfer proteins perform different functions in the cell.

The membranes of the thermoacidophilic archaeon Sulfolobus metallicus exhibit an oxygen consumption activity of 0.5 nmol O2 min−1 mg−1, which is insensitive to rotenone, suggesting the presence of a type-II NADH dehydrogenase. Following this observation, the enzyme was purified from solubilised membranes and characterised. The pure protein is a monomer with an apparent molecular mass of 49 kDa, having a high N-terminal amino acid sequence similarity towards other prokaryotic enzymes of the same type. It contains a covalently attached flavin, which was identified as being FMN by 31P-NMR spectroscopy, a novelty among type-II NADH dehydrogenases. Metal analysis showed the absence of iron, indicating that no FeS clusters are present in the protein. The average reduction potential of the FMN group was determined to be +160 mV, at 25 °C and pH 6.5, by redox titrations monitored by visible spectroscopy. Catalytically, the enzyme is a NADH:quinone oxidoreductase, as it is capable of transferring electrons from NADH to several quinones, including ubiquinone-1, ubiquinone-2 and caldariella quinone. Maximal turnover rates of 195 μmol NADH oxidized min−1 mg−1 at 60 °C were obtained using ubiquinone-2 as electron acceptor, after enzyme dilution and incubation with phospholipids.

The thermoacidophilic archaeon Acidianus ambivalens contains a monomeric 47 kDa type-II NADH dehydrogenase (NDH), which contains a covalently bound flavin. In this work, by a combination of several methods, namely 31P-nuclear magnetic resonance and fluorescence spectroscopies, it is proven that this enzyme contains covalent FMN, a novelty among this family of enzymes, which were so far thought to mainly have the flavin dinucleotide form. Discrimination between several possible covalent flavin linkages was achieved by spectral and fluorescence experiments, which identified an 8α-N(1)-histidylflavin-type of linkage. Analysis of the gene-deduced amino acid sequence of type-II NDH showed no transmembranar helices and allowed the definition of putative dinucleotide and quinone binding motifs. Further, it is suggested that membrane anchoring can be achieved via amphipatic helices.

Exoelectrogenic microorganisms are in the spotlight due to their unique respiratory mechanisms and potential applications in distinct biotechnological fields, including bioremediation, bioenergy production and microbial electrosynthesis. These applications rely on the capability of these microorganisms to perform extracellular electron transfer, a mechanism that allows the bacteria to transfer electrons to the cell’s exterior by establishing functional interfaces between different multiheme cytochromes at the inner membrane, periplasmic space, and outer membrane. The multiheme cytochrome CbcL from Geobacter sulfurreducens is associated to the inner membrane and plays an essential role in the transfer of electrons to final electron acceptors with a low redox potential, as Fe(III) oxides and electrodes poised at −100 mV. CbcL has a transmembranar di-heme b-type cytochrome domain with six helices, linked to a periplasmic cytochrome domain with nine c-type heme groups. The complementary usage of ultraviolet-visible, circular dichroism and nuclear magnetic resonance permitted the structural and functional characterization of CbcL’s periplasmic domain. The protein was found to have a high percentage of disordered regions and its nine hemes are low-spin and all coordinated by two histidine residues. The apparent midpoint reduction potential of the CbcL periplasmic domain was determined, suggesting a thermodynamically favorable transfer of electrons to the putative redox partner in the periplasm − the triheme cytochrome PpcA. The establishment of a redox complex between the two proteins was confirmed by probing the electron transfer reaction and the molecular interactions between CbcL and PpcA. The results obtained show for the first time how electrons are injected into the periplasm of Geobacter sulfurreducens for subsequent transfer to the cell’s exterior.

The extracellular electron transfer metabolism of Geobacter sulfurreducens is sustained by several multiheme c-type cytochromes. One of these is the dodecaheme cytochrome GSU1996 that belongs to a new sub-class of c-type cytochromes. GSU1996 is composed by four similar triheme domains (A-D). The C-terminal half of the molecule encompasses the domains C and D, which are connected by a small linker and the N-terminal half of the protein contains two domains (A and B) that form one structural unit. It was proposed that this protein works as an electrically conductive device in Geobacter sulfurreducens, transferring electrons within the periplasm or to outer-membrane cytochromes. In this work, a novel strategy was applied to characterize in detail the thermodynamic and kinetic properties of the hexaheme fragment CD of GSU1996. This characterization revealed the electron transfer process of GSU1996 for the first time, showing that a heme at the edge of the C-terminal of the protein is thermodynamic and kinetically competent to receive electrons from physiological redox partners. This information contributes towards understanding how this new sub-class of cytochromes functions as nanowires, and also increases the current knowledge of the extracellular electron transfer mechanisms in Geobacter sulfurreducens.