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The multiheme cytochrome MtrA enables microbial respiration by transferring electrons across the outer membrane to extracellular electron acceptors. While structural studies have identified residues that mediate MtrA binding to hemes and to other cytochromes that facilitate extracellular electron transfer (EET), the relative importance of these interactions for EET is not known. To better understand EET, we evaluated how insertion of an octapeptide across all MtrA backbone locations affects Shewanella oneidensis MR-1 respiration on Fe(III). EET efficiency was found to be inversely correlated with insertion proximity to the heme prosthetic groups. Mutants with decreased EET also arose from insertions in a subset of the regions that make residue-residue contacts with the porin MtrB, while all sites contacting the extracellular MtrC presented high peptide insertion tolerance. MtrA variants having peptide insertions within the CXXCH motifs that coordinate heme cofactors retained some ability to support respiration on Fe(III), although these variants presented significantly decreased EET. Furthermore, the fitness of cells expressing different MtrA variants under Fe(III)-respiring conditions correlated with anode reduction. The peptide-insertion profile, which represents the first comprehensive sequence-structure-function map for a multiheme cytochrome, implicates MtrA as a strategic protein engineering target for regulating EET.

The enzyme nicotine oxidoreductase (NicA2) is a member of the flavoprotein amine oxidase family that uses a cytochrome c protein (CycN) as its oxidant instead of dioxygen, which is the oxidant used by most other members of this enzyme family. We recently identified a potential binding site for CycN on the surface of NicA2 through rigid body docking [J. Biol. Chem. (2022) 298(8), 102251]. However, this potential binding interface has not been experimentally validated. In this paper, we used unnatural amino acid incorporation to probe the binding interface between NicA2 and CycN. Our results are consistent with a structural model of the NicA2-CycN complex predicted by protein-protein docking and AlphaFold, suggesting that this is the binding site for CycN on NicA2’s surface. Based on additional mutagenesis of potentially redox active residues in NicA2, we propose that electron transfer from NicA2’s flavin to CycN’s heme occurs without the assistance of a protein-derived wire.

Metals can play key roles in stabilizing protein structures, but ensuring their proper incorporation is a challenge when a metalloprotein is overexpressed in a non-native cellular environment. Here, we have used computational protein design tools to redesign cytochrome b(562) (cyt b(562)), which relies on the binding of its heme cofactor to achieve its proper fold, into a stable, heme-free protein with high structural similarity. The resulting protein, ApoCyt, features only four mutations and no metal-ligand or covalent bonds, yet has improved stability over cyt b(562). Mutagenesis studies and X-ray crystal structures reveal that the increase in stability is due to the computationally prescribed mutations, which stabilize the protein fold through a combination of hydrophobic packing interactions, hydrogen bonds, and cation-p interactions. Upon installation of the relevant mutations, ApoCyt is capable of assembling into previously reported, cytochrome-based trimeric and tetrameric assemblies, demonstrating that ApoCyt retains the structure and assembly properties of cyt b(562). The successful design of ApoCyt therefore enables further functional diversification of cytochrome-based assemblies and demonstrates that structural metal cofactors can be replaced by a small number of well-designed, non-covalent interactions.

In the past, many intensive attempts failed to capture or underestimated the copopulated intermediate conformers from the protein folding/unfolding reaction. We report a promising approach to kinetically trap, resolve, and quantify protein conformers that evolve during unfolding in solution. We conducted acid-induced unfolding of three model proteins (cytochrome

Cytochrome c(2) (cyt. c(2)) is a major element in electron transfer between redox proteins in bioenergetic membranes. While the interaction between cyt. c(2) and anionic lipids abundant in bioenergetic membranes has been reported, their effect on the shuttling activity of cyt. c(2) remains elusive. Here, the effect of anionic lipids on interaction and binding of cyt. c(2) to cytochrome bc(1) complex (bc(1)) is investigated using a combination of molecular dynamics (MD) and Brownian dynamics (BD) simulations. MD is used to generate thermally accessible conformations of cyt. c(2) and membrane-embedded bc(1), which were subsequently used in multi-replica BD simulations of cyt. c(2) diffusion from solution to bc(1), in the presence of various lipids. We show that, counterintuitively, anionic lipids facilitate association of cyt. c(2) to bc(1) by localizing its diffusion to the membrane surface. The observed lipid-mediated bc(1)-association is further enhanced by the oxidized state of cyt. c(2) in line with its physiological function. This lipid-mediated enhancement is salinity-dependent, and anionic lipids can disrupt cyt. c(2) - bc(1) interaction at non-physiological levels of salt. Our data highlight the importance of the redox state of cyt. c(2), the lipid composition of the chromatophore membrane, and the salinity of the chromatophore in regulating the efficiency of the electron shuttling process mediated by cyt. c(2). The conclusions can be extrapolated to mitochondrial systems and processes, or any bioenergetic membrane, given the structural similarity between cyt. c(2) and bc(1) and their mitochondrial counterparts.

Rhodopseudomonas palustris cytochrome c', a four-helix bundle, and the second ubiquitin-associated domain, UBA(2), a three-helix bundle from the human homologue of yeast Rad23, HHR23A, deviate from random coil behavior under denaturing conditions in a fold-specific manner. The random coil deviations in each of these folds occur near interhelical turns and loops in their tertiary structures. Here, we examine an additional three-helix bundle with an identical fold to UBA(2), but a highly divergent sequence, the first ubiquitin-associated domain, UBA(1), of HHR23A. We use histidine-heme loop formation methods, employing eight single histidine variants, to probe for denatured state conformational bias of a UBA(1) domain fused to the N-terminus of iso-1-cytochrome c (iso-1-Cytc). Guanidine hydrochloride (GuHCl) denaturation shows that the iso-1-Cytc domain unfolds first, followed by the UBA(1) domain. Denatured state (4 and 6 M GuHCl) histidine-heme loop formation studies show that as the size of the histidine-heme loop increases, loop stability decreases, as expected for the Jacobson-Stockmayer relationship. However, loops formed with His35, His31, and His15, of UBA(1), are 0.6-1.1 kcal/mol more stable than expected from the Jacobson-Stockmayer relationship, confirming the importance of deviations of the denatured state from random coil behavior near interhelical turns of helical domains for facilitating folding to the correct topology. For UBA(1) and UBA(2), hydrophobic clusters on either side of the turns partially explain deviations from random coil behavior; however, helix capping also appears to be important.