Biology subjects

Guberman-Pfeffer, M. J.

Publications and source records attributed to Guberman-Pfeffer, M. J..

7 recordsLinked to original sources

bioRxiv2024

Cytochrome 'Nanowires' are Physically Limited to Sub-Picoamp Currents that Suffice for Cellular Respiration

Mineral-respiring microorganisms from hydrothermal vents to terrestrial soils express filaments that electrically connect intracellular respiration to extracellular geochemistry. Filaments dubbed "cytochrome nanowires" (CNs) have been resolved by CryoEM, but whether they are the two-decades-long sought-after physiological nanowires remains unproven. To assess their functional competence, we analyzed biological redox conduction in all CNs by computing driving forces in the presence of redox anti-cooperativities, reorganization energies with electronic polarizability, and Marcus rates for diffusive and protein-limited flux models. The chain of heme cofactors in any CN must be densely packed to realize weak ([≤]0.01 eV) electronic coupling for electron transfer, as evidenced by a single Soret band produced from coincidental absorptions on multiple hemes. Dense packing, in turn, has three consequences: (1) limited driving forces ([≤]|0.3| eV) due to shared electrostatic microenvironments, (2) strong ([≤]0.12 eV) redox anti-cooperativities that would accentuate the free energy landscape if the linear heme arrangement did not dictate a contra-thermodynamic oxidation order, and (3) an entropic penalty that is offset by thioether tethers of the hemes to the protein backbone. These linkages physically necessitate the rate-throttling T-stacked motif (10-fold slower than the other highly conserved slip-stacked motif). If the sequence of slip- and T-stacked hemes in the CNs had the fastest known nanosecond rates at every step, a micron-long filament would carry a diffusive 0.02 pA current at a physiological 0.1 V, or a protein-limited current of 0.2 pA. Actual CNs have sub-optimal ([≤]102-fold lower), but sufficient conductivities for cellular respiration, with at most thousands of filaments needed for total cellular metabolic flux. Since cells likely discharge less than the 1.0 pA assumed here, and there are multiple pathways besides CNs for expelling electrons, the micro-to-milli-Siemens/cm conductivities are more than sufficient. Reported conductivities once used to argue for metallic-like pili against the cytochrome hypothesis and now illogically attributed to CNs remain inconsistent by 102-105-fold with the physical constraints imposed on biological redox conduction through multiheme architectures.

biophysics↗

bioRxiv2024

Five Cytochrome 'Nanowires' Suffice for Prokaryotes to Breathe Rocks Microns Away

Micron-scale electron transfer through polymeric cytochrome nanowires powers prokaryotic life from hydrothermal vents to terrestrial soils in ways not fully understood. Herein, six reduction potentials from recently reported spectroelectrochemistry are each assigned with <0.04 eV to the cryogenic electron microscopy structure of the hexa-heme homopolymeric outer-membrane cytochrome type S (OmcS) from Geobacter sulfurreducens using hybrid quantum/classical computations. The unambiguous assignments define a reversible free energy roller-coaster that is dynamically modulated by <0.1 V under the flow of electrons due to redox cooperativities between adjacent hemes. A physiologically relevant tens to hundreds of filaments are predicted to suffice for cellular respiration by pairing, in the context of non-adiabatic Marcus theory, the free energy landscape with reorganization energies that account for active site or protein-water electronic polarizability, and electronic couplings characteristic of the highly conserved heme packing motifs. General considerations on protein electron transfer and comparison to all known cytochrome nanowires suggest the mechanistic insights are broadly applicable to multi-heme cytochromes in all kingdoms of life.

biophysics↗

bioRxiv2024

Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to unpick the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

biochemistry↗

bioRxiv2023

Are Electrical Characterizations Consistent with the Cytochrome Structures of Geobacter 'Nanowires'

Electrically conductive filaments from Geobacter sulfurreducens were reported to be pili with metallic-like conductivity, and yet were later shown to be redox-active cytochromes by cryogenic electron microscopy. It has recently been argued that the filaments were simply misidentified, implying that key observations formerly used to refute the involvement of cytochromes in conductivity now must be ascribed to them. Herein, the temperature, pH, voltage, crystallinity, charge propagation, and aromatic density-related dependencies of the conductivity reported for putative pili are re-examined in light of the CryoEM structures of cytochrome filaments. It is demonstrated that: O_LIElectrons flow through cytochrome filaments in a succession of redox reactions for which the energetics are physically constrained and the kinetics are largely independent of protein identity for highly conserved heme packing geometries. Computed heme-to-heme electron transfer rates in cytochrome filaments agree, on average, within a factor of 10 of rates experimentally determined in other multi-heme proteins with the same heme packing geometries. C_LIO_LIT-stacked heme pairs, which comprise nearly or exactly half of all heme pairs in cytochrome filaments are electronic coupling-constrained bottlenecks for electron transfer that set the rate-limiting reaction to the {micro}s timescale, which is fast enough compared to typical ms enzymatic turnover. Tuning the conductivity of cytochromes over the reported [~]107-fold range for filaments from G. sulfurreducens strains with pili variants seems both physically implausible and physiologically irrelevant if those filaments are supposed to be cytochromes. C_LIO_LIThe protein-limited flux for redox conduction through a 300-nm filament of T- and slip-stacked heme pairs is predicted to be [~]0.1 pA; a G. sulfurreducens cell discharging [~]1 pA/s would need at least 10 filaments, which is consistent with experimental estimates of filament abundance. The experimental currents for the Omc- S and Z filaments at a physiologically relevant 0.1 V bias, however, are [~]10 pA and [~]10 nA, respectively. Some of the discrepancy is attributable to the experimental conditions of a dehydrated protein adsorbed on a bear Au- electrode that contacts [~]102 hemes, and in the case of conducting probe atomic force microscopy, is crushed under forces known to deform and change the electron transport mechanism through more highly-structured proteins. C_LIO_LIPreviously observed hallmarks of synthetic organic metallic-like conductivity ascribed to pili are inconsistent with the structurally resolved cytochrome filaments under physiological conditions, including (I) increased crystallinity promoting electron delocalization, (II) carbon nanotube-like charge propagation, and (III) an exponential increase-then-decrease in conductivity upon cooling, which was only explain by a model predicted on redox potentials known to be experimentally false. Furthermore, spectroscopic structural characterizations of OmcZ that attest to a huge acid-induced transition to a more crystalline state enhancing conductivity either strongly disagree with CryoEM analyses at higher pH values or give inconclusive results that can be overly interpreted. C_LI Overall, a significant discrepancy currently exists--not between theory and experiment--but between the CryoEM cytochrome filament structure in one hand and the other functional characterizations of Geobacter nanowires in the other. The CryoEM structures, theoretical models, biological experiments, and kinetic analyses are all in agreement about the nature and rate of electron transfer in multi-heme architectures under physiological conditions, and stand opposed to the solid-state functional characterizations of Geobacter filaments reported to date. The physiological relevance and/or physical plausibility of some experiments should be examined further.

biophysics↗

bioRxiv2023

From Hot Water to Dry Dirt: Microbes Use Cytochrome 'Nanowires' of Similar Conductivity but Different Structure

Micron-scale electron transfer through polymeric cytochrome nanowires powers prokaryotic life from hydrothermal vents to terrestrial soils in ways not fully understood. How much structural diversity optimizes electrical conductivity for survival in these different habitats is challenging to assess experimentally. Herein, physiologically relevant redox conduction is computationally assessed in cytochrome filaments from Geobacter sulfurreducens (OmcE, OmcS, and OmcZ), Pyrobaculum calidifontis (A3MW92), and Archaeoglobus veneficus (F2KMU8). A newly implemented Python program, BioDC, is used and validated against redox currents predicted from considerably more expensive molecular dynamics and quantum mechanical/molecular mechanical calculations. BioDC uses the heme solvent accessibility, stacking geometry, and redox-linked change in electrostatic energy to estimate electron transfer energetics. Leveraging this efficiency, structurally diverse cytochrome nanowires from different organisms are shown to have similar redox conductivities. A functionally robust heme chain packaged in habitat-customized proteins is proposed to be a general evolutionary design principle for cytochrome nanowires widely distributed among prokaryotes. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=198 HEIGHT=200 SRC="FIGDIR/small/544705v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@ac61d9org.highwire.dtl.DTLVardef@14c2e87org.highwire.dtl.DTLVardef@82d60corg.highwire.dtl.DTLVardef@1b2cbd6_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Structural Determinants of Redox Conduction Favor Robustness over Tunability in Microbial Cytochrome Nanowires

Helical homopolymers of multiheme cytochromes catalyze biogeochemically significant electron transfers with a reported 103-fold variation in conductivity. Herein, classical molecular dynamics and hybrid quantum/classical molecular mechanics are used to elucidate the structural determinants of the redox potentials and conductivities of the tetra-, hexa-, and octaheme outer-membrane cytochromes E, S, and Z, respectively, from Geobacter sulfurreducens. Second-sphere electrostatic interactions acting on minimally polarized heme centers are found to regulate redox potentials over a computed 0.5-V range. However, the energetics of redox conduction are largely robust to the structural diversity: Single-step electronic couplings ([<]Hmn[>]), reaction free energies [Formula], and reorganization energies ({lambda}mn) are always respectively <|0.026|, <|0.26|, and between 0.5 - 1.0 eV. With these conserved parameter ranges, redox conductivity differed by less than a factor of 10 among the nanowires and is sufficient to meet the demands of cellular respiration if 102 - 103 nanowires are expressed. The nanowires are proposed to be differentiated by the protein packaging to interface with a great variety of environments, and not by conductivity, because the rate-limiting electron transfers are elsewhere in the respiratory process. Conducting-probe atomic force microscopy measurements that find conductivities 103-106-fold more than cellular demands are suggested to report on functionality that is either not used or not accessible under physiological conditions. The experimentally measured difference in conductivity between Omc- S and Z is suggested to not be an intrinsic feature of the CryoEM-resolved structures.

biophysics↗

bioRxiv2022

Heme Hopping Falls Short: What Explains Anti-Arrhenius Conductivity in a Multi-heme Cytochrome Nanowire?

A helical homopolymer of the outer-membrane cytochrome type S (OmcS) was proposed to electrically connect a common soil bacterium, Geobacter sulfurreducens, with minerals and other microbes for biogeochemically important processes. OmcS exhibits a surprising rise in conductivity upon cooling from 300 to 270 K that has recently been attributed to a restructuring of H-bonds, which in turn modulates heme redox potentials. This proposal is more thoroughly examine herein by (1) analyzing H-bonding at 13 temperatures encompassing the entire experimental range; (2) computing redox potentials with quantum mechanics/molecular mechanics for 10-times more (3000) configurations sampled from 3-times longer (2 s) molecular dynamics, as well as 3 s of constant redox and pH molecular dynamics; and (3) modeling redox conduction with both single-particle diffusion and multi-particle flux kinetic schemes. Upon cooling by 30 K, the connectivity of the intra-protein H-bonding network was highly (86%) similar. An increase in the density and static dielectric constant of the filaments hydration shell caused a -0.002 V/K shift in heme redox potentials, and a factor of 2 decrease in charge mobility. Revision of a too-far negative redox potential in prior work (-0.521 V; expected = -0.350 - +0.150 V; new Calc. = -0.214 V vs. SHE) caused the mobility to be greater at high versus low temperature, opposite to the original prediction. These solution-phase redox conduction models failed to reproduce the experimental conductivity of electrode-absorbed, partially dehydrated, and possibly aggregated OmcS filaments. Some improvement was seen by neglecting reorganization energy from the solvent to model dehydration. Correct modeling of the physical state is suggested to be a prerequisite for reaching a verdict on the operative charge transport mechanism and the molecular basis of its temperature response.

biophysics↗