Whole-cell circular dichroism difference spectroscopy reveals an in vivo-specific deca-heme conformation in bacterial surface cytochromes.
ABSTRACT: We established whole-cell circular dichroism difference spectroscopy to identify the inter-heme interaction in deca-heme cytochrome protein MtrC in whole cell. Our data showed that the heme alignment of reduced MtrC in whole cell is distinct from that in purified one, suggesting the in vivo specific electron transport kinetics.
Project description:Certain dissimilatory bacteria have the remarkable ability to use extracellular metal oxide minerals instead of oxygen as terminal electron sinks, using a process known as "extracellular respiration". Specialized multiheme cytochromes located on the outer membrane of the microbe were shown to be crucial for electron transfer from the cell surface to the mineral. This process is facilitated by soluble, biogenic flavins secreted by the organism for the purpose of acting as an electron shuttle. However, their interactions with the outer-membrane cytochromes are not established on a molecular scale. Here, we study the interaction between the outer-membrane deca-heme cytochrome MtrC from Shewanella oneidensis and flavin mononucleotide (FMN in fully oxidized quinone form) using computational docking. We find that interaction of FMN with MtrC is significantly weaker than with known FMN-binding proteins, but identify a mildly preferred interaction site close to heme 2 with a dissociation constant (Kd) = 490 ?M, in good agreement with recent experimental estimates, Kd = 255 ?M. The weak interaction with MtrC can be qualitatively explained by the smaller number of hydrogen bonds that the planar headgroup of FMN can form with this protein compared to FMN-binding proteins. Molecular dynamics simulation gives indications for a possible conformational switch upon cleavage of the disulphide bond of MtrC, but without concomitant increase in binding affinities according to this docking study. Overall, our results suggest that binding of FMN to MtrC is reversible and not highly specific, which may be consistent with a role as redox shuttle that facilitates extracellular respiration.
Project description:Multi-heme cytochrome c (Cytc) proteins are key for transferring electrons out of cells, to enable intracellular oxidation to proceed in the absence of O2. In these proteins most of the hemes are arranged in a linear array suggesting a facile path for electronic conduction. To test this, we studied solvent-free electron transport across two multi-heme Cytc-type proteins: MtrF (deca-heme Cytc) and STC (tetra-heme Cytc). Transport is measured across monolayers of these proteins in a solid state configuration between Au electrodes. Both proteins showed 1000× higher conductance than single heme, or heme-free proteins, but similar conductance to monolayers of conjugated organics. Conductance is found to be temperature-independent (320-80 K), suggesting tunneling as the transport mechanism. This mechanism is consistent with I-V curves modelling, results of which could be interpreted by having protein-electrode coupling as rate limiting, rather than transport within the proteins.
Project description:The naturally widespread process of electron transfer from metal reducing bacteria to extracellular solid metal oxides entails unique biomolecular machinery optimized for long-range electron transport. To perform this function efficiently, microorganisms have adapted multiheme c-type cytochromes to arrange heme cofactors into wires that cooperatively span the cellular envelope, transmitting electrons along distances greater than 100 Å. Implications and opportunities for bionanotechnological device design are self-evident. However, at the molecular level, how these proteins shuttle electrons along their heme wires, navigating intraprotein intersections and interprotein interfaces efficiently, remains a mystery thus far inaccessible to experiment. To shed light on this critical topic, we carried out extensive quantum mechanics/molecular mechanics simulations to calculate stepwise heme-to-heme electron transfer rates in the recently crystallized outer membrane deca-heme cytochrome MtrF. By solving a master equation for electron hopping, we estimate an intrinsic, maximum possible electron flux through solvated MtrF of 10(4)-10(5) s(-1), consistent with recently measured rates for the related multiheme protein complex MtrCAB. Intriguingly, our calculations show that the rapid electron transport through MtrF is the result of a clear correlation between heme redox potential and the strength of electronic coupling along the wire: thermodynamically uphill steps occur only between electronically well-connected stacked heme pairs. This observation suggests that the protein evolved to harbor low-potential hemes without slowing down electron flow. These findings are particularly profound in light of the apparently well-conserved staggered cross-heme wire structural motif in functionally related outer membrane proteins.
Project description:A number of species of Gram-negative bacteria can use insoluble minerals of Fe(III) and Mn(IV) as extracellular respiratory electron acceptors. In some species of Shewanella, deca-heme electron transfer proteins lie at the extracellular face of the outer membrane (OM), where they can interact with insoluble substrates. To reduce extracellular substrates, these redox proteins must be charged by the inner membrane/periplasmic electron transfer system. Here, we present a spectro-potentiometric characterization of a trans-OM icosa-heme complex, MtrCAB, and demonstrate its capacity to move electrons across a lipid bilayer after incorporation into proteoliposomes. We also show that a stable MtrAB subcomplex can assemble in the absence of MtrC; an MtrBC subcomplex is not assembled in the absence of MtrA; and MtrA is only associated to the membrane in cells when MtrB is present. We propose a model for the modular organization of the MtrCAB complex in which MtrC is an extracellular element that mediates electron transfer to extracellular substrates and MtrB is a trans-OM spanning beta-barrel protein that serves as a sheath, within which MtrA and MtrC exchange electrons. We have identified the MtrAB module in a range of bacterial phyla, suggesting that it is widely used in electron exchange with the extracellular environment.
Project description:Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O2 or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the ? subunits. However, these changes are not observed in the isolated ? chain (?4 homotetramer) and, therefore, are associated with the ?-? inter-subunit interactions. For the communications between ? and ? subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O2 binding mechanism of Hb A.
Project description:The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice.We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured.Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450.In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism.
Project description:Preparation of heme model complexes is a challenging subject of long-standing interest for inorganic chemists. His93Gly sperm whale myoglobin (H93G Mb) has the proximal His replaced with the much smaller non-coordinating Gly. This leaves a cavity on the proximal side of the heme into which a wide variety of exogenous ligands can be delivered. The end result is a remarkably versatile scaffold for the preparation of model heme adducts to mimic the heme iron coordination structure of native heme proteins. In this review, we first summarize the quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of the H93G Mb cavity mutant that facilitates the preparation of mixed-ligand derivatives. Then we review our use of magnetic circular dichroism and electronic absorption spectroscopy to characterize nitrogen-, oxygen-, and sulfur-donor-ligated H93G Mb adducts with an emphasis on species not easily prepared by other heme model system approaches and those that serve as spectroscopic models for native heme proteins.
Project description:The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII -dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.
Project description:It is generally accepted that the inactive P420 form of cytochrome P450 (CYP) involves the protonation of the native cysteine thiolate to form a neutral thiol heme ligand. On the other hand, it has also been suggested that recruitment of a histidine to replace the native cysteine thiolate ligand might underlie the P450 ? P420 transition. Here, we discuss resonance Raman investigations of the H93G myoglobin (Mb) mutant in the presence of tetrahydrothiophene (THT) or cyclopentathiol (CPSH), and on pressure-induced cytochrome P420cam (CYP101), that show a histidine becomes the heme ligand upon CO binding. The Raman mode near 220 cm?¹, normally associated with the Fe-histidine vibration in heme proteins, is not observed in either reduced P420cam or the reduced H93G Mb samples, indicating that histidine is not the ligand in the reduced state. The absence of a mode near 220 cm?¹ is also inconsistent with a generalization of the suggestion that the 221 cm?¹ Raman mode, observed in the P420-CO photoproduct of inducible nitric oxide synthase (iNOS), arises from a thiol-bound ferrous heme. This leads us to assign the 218 cm?¹ mode observed in the 10 ns P420cam-CO photoproduct Raman spectrum to a Fe-histidine vibration, in analogy to many other histidine-bound heme systems. Additionally, the inverse correlation plots of the ?Fe-His and ?CO frequencies for the CO adducts of P420cam and the H93G analogs provide supporting evidence that histidine is the heme ligand in the P420-CO-bound state. We conclude that, when CO binds to the ferrous P420 state, a histidine ligand is recruited as the heme ligand. The common existence of an HXC-Fe motif in many CYP systems allows the C ? H ligand switch to occur with only minor conformational changes. One suggested conformation of P420-CO involves the addition of another turn in the proximal L helix so that, when the protonated Cys ligand is dissociated from the heme, it can become part of the helix, and the heme is ligated by the His residue from the adjoining loop region. In other systems, such as iNOS and CYP3A4 (where the HXC-Fe motif is not found), a somewhat larger conformational change would be necessary to recuit a nearby histidine.
Project description:The polypeptide and structural gene for a high-molecular-mass c-type cytochrome, cytochrome c553O, was isolated from the methanotroph Methylococcus capsulatus Bath. Cytochrome c553O is a homodimer with a subunit molecular mass of 124,350 Da and an isoelectric point of 6. 0. The heme c concentration was estimated to be 8.2 +/- 0.4 mol of heme c per subunit. The electron paramagnetic resonance spectrum showed the presence of multiple low spin, S = 1/2, hemes. A degenerate oligonucleotide probe synthesized based on the N-terminal amino acid sequence of cytochrome c553O was used to identify a DNA fragment from M. capsulatus Bath that contains occ, the gene encoding cytochrome c553O. occ is part of a gene cluster which contains three other open reading frames (ORFs). ORF1 encodes a putative periplasmic c-type cytochrome with a molecular mass of 118, 620 Da that shows approximately 40% amino acid sequence identity with occ and contains nine c-heme-binding motifs. ORF3 encodes a putative periplasmic c-type cytochrome with a molecular mass of 94, 000 Da and contains seven c-heme-binding motifs but shows no sequence homology to occ or ORF1. ORF4 encodes a putative 11,100-Da protein. The four ORFs have no apparent similarity to any proteins in the GenBank database. The subunit molecular masses, arrangement and number of hemes, and amino acid sequences demonstrate that cytochrome c553O and the gene products of ORF1 and ORF3 constitute a new class of c-type cytochrome.