Bruno M. Fonseca

Mind the gap: cytochrome interactions reveal electron pathways across the periplasm of Shewanella oneidensis MR-1

Extracellular electron transfer is the key metabolic trait that enables some bacteria to play a significant role in the biogeochemical cycling of metals and in bioelectrochemical devices such as microbial fuel cells. In Shewanella oneidensis MR-1, electrons generated in the cytoplasm by catabolic processes must cross the periplasmic space to reach terminal oxidoreductases found at the cell surface. Lack of knowledge on how these electrons flow across the periplasmic space is one of the unresolved issues related with extracellular electron transfer. Using NMR to probe protein-protein interactions, kinetic measurements of electron transfer and electrostatic calculations, we were able to identify protein partners and their docking sites, and determine the dissociation constants. The results showed that both STC (small tetrahaem cytochrome c) and FccA (flavocytochrome c) interact with their redox partners, CymA and MtrA, through a single haem, avoiding the establishment of stable redox complexes capable of spanning the periplasmic space. Furthermore, we verified that the most abundant periplasmic cytochromes STC, FccA and ScyA (monohaem cytochrome c5) do not interact with each other and this is likely to be the consequence of negative surface charges in these proteins. This reveals the co-existence of two non-mixing redox pathways that lead to extracellular electron transfer in S. oneidensis MR-1 established through transient protein interactions.

Exploring the molecular mechanisms of electron shuttling across the microbe/metal space

Dissimilatory metal reducing organisms play key roles in the biogeochemical cycle of metals as well as in the durability of submerged and buried metallic structures. The molecular mechanisms that support electron transfer across the microbe-metal interface in these organisms remain poorly explored. It is known that outer membrane proteins, in particular multiheme cytochromes, are essential for this type of metabolism, being responsible for direct and indirect, via electron shuttles, interaction with the insoluble electron acceptors. Soluble electron shuttles such as flavins, phenazines, and humic acids are known to enhance extracellular electron transfer. In this work, this phenomenon was explored. All known outer membrane decaheme cytochromes from Shewanella oneidensis MR-1 with known metal terminal reductase activity and a undecaheme cytochrome from Shewanella sp. HRCR-6 were expressed and purified. Their interactions with soluble electron shuttles were studied using stopped-flow kinetics, NMR spectroscopy, and molecular simulations. The results show that despite the structural similarities, expected from the available structural data and sequence homology, the detailed characteristics of their interactions with soluble electron shuttles are different. MtrC and OmcA appear to interact with a variety of different electron shuttles in the close vicinity of some of their hemes, and with affinities that are biologically relevant for the concentrations typical found in the medium for this type of compounds. All data support a view of a distant interaction between the hemes of MtrF and the electron shuttles. For UndA a clear structural characterization was achieved for the interaction with AQDS a humic acid analog. These results provide guidance for future work of the manipulation of these proteins toward modulation of their role in metal attachment and reduction.

The role of intramolecular interactions in the functional control of multiheme cytochromes c

Detailed thermodynamic and structural data measured in soluble monomeric multiheme cytochromes c provided the basis to investigate the functional significance of interactions between redox co‐factors. The steep decay of intramolecular interactions with distance means that close proximity of the redox centers is necessary to modulate the intrinsic reduction potentials in a significant way. This ensures selection of specific populations during redox activity in addition to maintaining fast intramolecular electron transfer. Therefore, intramolecular interactions between redox co‐factors play an important role in establishing the biological function of the protein by controlling how electrons flow through and are distributed among the co‐factors.

Exploration of the ‘cytochromome’ of Desulfuromonas acetoxidans, a marine bacterium capable of powering microbial fuel cells

Recent progress in bacterial genomic analysis has revealed a vast number of genes that encode c-type cytochromes that contain multiple heme cofactors. This high number of multiheme cytochromes in several bacteria has been correlated with their great respiratory flexibility, and in what concerns biotechnological applications, has been correlated with electricity production in Microbial Fuel Cells. Desulfuromonas acetoxidans, a member of the Geobactereaceae family, is one of these organisms for which the genome was recently made available, coding for 47 putative multiheme cytochromes. The growth of D. acetoxidans in different media allowed the identification of the cytochromes dominant in each condition. The triheme cytochrome c(7) is always present suggesting a key role in the bioenergetic metabolism of this organism, and a dodecaheme cytochrome of low homology with other proteins in the databases was also isolated. Different cytochromes are found for different growth conditions showing that their roles can be assigned to specific bioenergetic electron transfer routes.

Characterization of the periplasmic redox network that sustains the versatile anaerobic metabolism of Shewanella oneidensis MR-1

The versatile anaerobic metabolism of the Gram-negative bacterium Shewanella oneidensis MR-1 (SOMR-1) relies on a multitude of redox proteins found in its periplasm. Most are multiheme cytochromes that carry electrons to terminal reductases of insoluble electron acceptors located at the cell surface, or bona fide terminal reductases of soluble electron acceptors. In this study, the interaction network of several multiheme cytochromes was explored by a combination of NMR spectroscopy, activity assays followed by UV-visible spectroscopy and comparison of surface electrostatic potentials. From these data the small tetraheme cytochrome (STC) emerges as the main periplasmic redox shuttle in SOMR-1. It accepts electrons from CymA and distributes them to a number of terminal oxidoreductases involved in the respiration of various compounds. STC is also involved in the electron transfer pathway to reduce nitrite by interaction with the octaheme tetrathionate reductase (OTR), but not with cytochrome c nitrite reductase (ccNiR). In the main pathway leading the metal respiration STC pairs with flavocytochrome c (FccA), the other major periplasmic cytochrome, which provides redundancy in this important pathway. The data reveals that the two proteins compete for the binding site at the surface of MtrA, the decaheme cytochrome inserted on the periplasmic side of the MtrCAB–OmcA outer-membrane complex. However, this is not observed for the MtrA homologues. Indeed, neither STC nor FccA interact with MtrD, the best replacement for MtrA, and only STC is able to interact with the decaheme cytochrome DmsE of the outer-membrane complex DmsEFABGH. Overall, these results shown that STC plays a central role in the anaerobic respiratory metabolism of SOMR-1. Nonetheless, the trans-periplasmic electron transfer chain is functionally resilient as a consequence of redundancies that arise from the presence of alternative pathways that bypass/compete with STC.

Binding of ligands originates small perturbations on the microscopic thermodynamic properties of a multicentre redox protein

NMR and visible spectroscopy coupled to redox measurements were used to determine the equilibrium thermodynamic properties of the four haems in cytochrome c3 under conditions in which the protein was bound to ligands, the small anion phosphate and the protein rubredoxin with the iron in the active site replaced by zinc. Comparison of these results with data for the isolated cytochrome shows that binding of ligands causes only small changes in the reduction potentials of the haems and their pairwise interactions, and also that the redox‐sensitive acid–base centre responsible for the redox–Bohr effect is essentially unaffected. Although neither of the ligands tested is a physiological partner of cytochrome c3, the small changes observed for the thermodynamic properties of cytochrome c3 bound to these ligands vs. the unbound state, indicate that the thermodynamic properties measured for the isolated protein are relevant for a physiological interpretation of the role of this cytochrome in the bioenergetic metabolism of Desulfovibrio.

Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins

Specific isotopic labeling of hemes provides a unique opportunity to characterize the structure and function of heme-proteins. Unfortunately, current methods do not allow efficient labeling in high yields of multiheme cytochromes c, which are of great biotechnological interest. Here, a method for production of recombinant multiheme cytochromes c in Escherichia coli with isotopically labeled hemes is reported. A small tetraheme cytochrome of 12 kDa from Shewanella oneidensis MR-1 was used to demonstrate the method, achieving a production of 4 mg pure protein per liter. This method achieves, in a single step, efficient expression and incorporation of hemes isotopically labeled in specific atom positions adequate for spectroscopic characterization of these complex heme proteins. It is, furthermore, of general application to heme proteins, opening new possibilities for the characterization of this important class of proteins.

A putative siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIMB 400: cloning, expression, purification, crystallization and X-ray diffraction analysis

The gene encoding a putative siderophore-interacting protein from the marine bacterium S. frigidimarina was successfully cloned, followed by expression and purification of the gene product. Optimized crystals diffracted to 1.35 Å resolution and preliminary crystallographic analysis is promising with respect to structure determination and increased insight into the poorly understood molecular mechanisms underlying iron acquisition.

Molecular mechanisms of heme based sensors from sediment organisms capable of extracellular electron transfer

Bioelectrochemical systems (BES) rely on the metabolism of sediment bacteria capable of forming electrogenic biofilms to generate electrical work. The environment across the thickness of the biofilm is variable and in order for the cells to maintain their viability they require molecular sensors that allow them to adapt their metabolism to their respective environment. The DcrA sensor from Desulfovibrio vulgaris and the GSU 0582 and 0935 sensor domains from Geobacter sulfurreducens appear to function as redox sensors. The SO2144 sensor domain from Shewanella oneidensis MR-1 and the cytochrome c″ from Methylophilus methylotrophus appear to function as NO sensors. Although M. methylotrophus is not known to colonize electrodes on BES, the characterization of cytochrome c″ serves to illustrate the general mechanism of NO sensing similar to that of the heme based sensors of sediment bacteria used in BES. In all cases, conformational changes initiated by the signal trigger the response. What appears to set these two groups of proteins apart is the poise of the heme in the sensors. In the case of redox sensors the hemes appear to be low spin iron II axially coordinated by two residues of the protein. In the case of NO sensors the heme appears to be high spin iron II with the distal coordination site vacant. Understanding of the molecular bases for signal and ligand discrimination may enable a fine control of biofilm formation in bioelectrochemical systems or the development of novel biosensors.

Corrigendum to “The role of intramolecular interactions in the functional control of multiheme cytochromes c” [FEBS Lett. 586 (2012) 504–509]

Fig. 2. Distance dependence of the pairwise interactions between the hemes. Squares illustrate data for the STC from the Shewanella genus [30,31]; Triangles illustrate data for the flavocytochromes c3 from the Shewanella genus [32]; Diamonds illustrate data for the cytochrome c7/Ppc from Desulfuromonas and Geobacter genera [24,25]; the rectangle illustrates the range of the interactions reported for the cytochrome c4 [33]; open circles illustrate data for the cytochromes c3 from the Desulfovibrio and Desulfomicrobium genera [27–29]. Distances were measured between iron atoms from the protein structures with the following PDB codes: 1M1Q; 2K3V; 1QJD; 1D4D; 1HH5; 2LDO; 3BXU; 3H4N; 3H34; 1RWJ; 1WAD; 2CTH; 1UPD; 2BQ4; 2CY3; 1W7O; 1M70, using the program Pymol v0.99. The solid line was obtained with a Debye–Hückel model of shielded electrostatic interactions considering and effective dielectric constant of 8.6 and Debye length of 7.7 Å [42].