David R. Benson

Heme Uptake and Metabolism in Bacteria

All but a few bacterial species have an absolute need for heme, and most are able to synthesize it via a pathway that is highly conserved among all life domains. Because heme is a rich source for iron, many pathogenic bacteria have also evolved processes for sequestering heme from their hosts. The heme biosynthesis pathways are well understood at the genetic and structural biology levels. In comparison, much less is known about the heme acquisition, trafficking, and degradation processes in bacteria. Gram-positive and Gram-negative bacteria have evolved similar strategies but different tactics for importing and degrading heme, likely as a consequence of their different cellular architectures. The differences are manifested in distinct structures for molecules that perform similar functions. Consequently, the aim of this chapter is to provide an overview of the structural biology of proteins and protein-protein interactions that enable Gram-positive and Gram-negative bacteria to sequester heme from the extracellular milieu, import it to the cytosol, and degrade it to mine iron.

Comparison of cytochromes b5 from insects and vertebrates.

We report a 1.55 Å X‐ray crystal structure of the heme‐binding domain of cytochrome b5 from Musca domestica (house fly; HF b5), and compare it with previously published structures of the heme‐binding domains of bovine microsomal cytochrome b5 (bMc b5) and rat outer mitochondrial membrane cytochrome b5 (rOM b5). The structural comparison was done in the context of amino acid sequences of all known homologues of the proteins under study. We show that insect b5s contain an extended hydrophobic patch at the base of the heme binding pocket, similar to the one previously shown to stabilize mammalian OM b5s relative to their Mc counterparts. The hydrophobic patch in insects includes a residue with a bulky hydrophobic side chain at position 71 (Met). Replacing Met71 in HF b5 with Ser, the corresponding residue in all known mammalian Mc b5s, is found to substantially destabilize the holoprotein. The destabilization is a consequence of two related factors: (1) a large decrease in apoprotein stability and (2) extension of conformational disruption in the apoprotein beyond the empty heme binding pocket (core 1) and into the heme‐independent folding core (core 2). Analogous changes have previously been shown to accompany replacement of Leu71 in rOM b5 with Ser. That the stabilizing role of Met71 in HF b5 is manifested primarily in the apo state is highlighted by the fact that its crystallographic Cα B factor is modestly larger than that of Ser71 in bMc b5, indicating that it slightly destabilizes local polypeptide conformation when heme is in its binding pocket. Finally, we show that the final unit of secondary structure in the cytochrome b5 heme‐binding domain, a 310 helix known as α6, differs substantially in length and packing interactions not only for different protein isoforms but also for given isoforms from different species. Proteins 2007.

Stabilizing roles of residual structure in the empty heme binding pockets and unfolded states of microsomal and mitochondrial apocytochrome b5

The microsomal (Mc) and mitochondrial (OM) isoforms of mammalian cytochrome b5 are the products of different genes, which likely arose via duplication of a primordial gene and subsequent functional divergence. Despite sharing essentially identical folds, heme‐polypeptide interactions are stronger in OM b5s than in Mc b5s due to the presence of two conserved patches of hydrophobic amino acid side chains in the OM heme binding pockets. This is of fundamental interest in terms of understanding heme protein structure–function relationships, because stronger heme–polypeptide interactions in OM b5s in comparison to Mc b5s may represent a key source of their more negative reduction potentials. Herein we provide evidence that interactions amongst the amino acid side chains contributing to the hydrophobic patches in rat OM (rOM) b5 persist when heme is removed, rendering the empty heme binding pocket of rOM apo‐b5 more compact and less conformationally dynamic than that in bovine Mc (bMc) apo‐b5. This may contribute to the stronger heme binding by OM apo‐b5 by reducing the entropic penalty associated with polypeptide folding. We also show that when bMc apo‐b5 unfolds it adopts a structure that is more compact and contains greater nonrandom secondary structure content than unfolded rOM apo‐b5. We propose that a more robust β‐sheet in Mc apo‐b5s compensates for the absence of the hydrophobic packing interactions that stabilize the heme binding pocket in OM apo‐b5s.

Accommodating a nonconservative internal mutation by water-mediated hydrogen bonding between β-sheet strands: a comparison of human and rat type B (mitochondrial) cytochrome b5.

Mammalian type B (mitochondrial) b(5) cytochromes exhibit greater amino acid sequence diversity than their type A (microsomal) counterparts, as exemplified by the type B proteins from human (hCYB5B) and rat (rCYB5B). The comparison of X-ray crystal structures of hCYB5B and rCYB5B reported herein reveals a striking difference in packing involving the five-strand β-sheet, which can be attributed to fully buried residue 21 in strand β4. The greater bulk of Leu21 in hCYB5B in comparison to that of Thr21 in rCYB5B results in a substantial displacement of the first two residues in β5, and consequent loss of two of the three hydrogen bonds between β5 and β4. Hydrogen bonding between the residues is instead mediated by two well-ordered, fully buried water molecules. In a 10 ns molecular dynamics simulation, one of the buried water molecules in the hCYB5B structure exchanged readily with solvent via intermediates having three water molecules sandwiched between β4 and β5. When the buried water molecules were removed prior to a second 10 ns simulation, β4 and β5 formed persistent hydrogen bonds identical to those in rCYB5B, but the Leu21 side chain was forced to adopt a rarely observed conformation. Despite the apparently greater ease of access of water to the interior of hCYB5B than of rCYB5B suggested by these observations, the two proteins exhibit virtually identical stability, dynamic, and redox properties. The results provide new insight into the factors stabilizing the cytochrome b(5) fold.

Thermodynamics of Carbon Monoxide Binding by Helical Hemoprotein Models: the Effect of a Competing Intramolecular Ligand

Abstract Ferrous hemoprotein models 1 and 2 exhibit bis-histidine/mono-histidine coordination equilibrium in aqueous solution. Carbon monoxide binds more tightly to 1 than to 2, a result of stronger Fe(II)–His coordination in 2 arising from interactions between the Trp side chain and the porphyrin ring. Coordination of the more weakly bound histidine ligands to Fe(II) in 1 is shown to be enthalpically favored but entropically disfavored due to the accompanying change in peptide conformation from random coil to α-helix. We demonstrate that competition from the intramolecular His ligand in 1 reduces ΔH° of CO binding compared to the mono-His coordinated form of the compound, an effect which is largely compensated by the positive entropy term due to unwinding of the peptide helix. Trading enthalpic stabilization of an Fe-ligand bond for an entropy gain due to a protein conformational change may be a common mode of action for hemoproteins which function as small molecule sensors.

House fly cytochrome b5 exhibits kinetically trapped hemin and selectivity in hemin binding.

We report that cytochrome b(5) (cyt b(5)) from Musca domestica (house fly) is more thermally stable than all other microsomal (Mc) cytochromes b(5) that have been examined to date. It also exhibits a much higher barrier to equilibration of the two isomeric forms of the protein, which differ by a 180 degrees rotation about the alpha-gamma-meso axis of hemin (ferric heme). In fact, hemin is kinetically trapped in a nearly statistical 1.2:1 ratio of rotational forms in freshly expressed protein. The equilibrium ratio (5.5:1) is established only upon incubation at temperatures above 37 degrees C. House fly Mc cyt b(5) is only the second b-hemoprotein that has been shown to exhibit kinetically trapped hemin at room temperature or above, the first being cyt b(5) from the outer membrane of rat liver mitochondria (rat OM cyt b(5)). Finally, we show that the small excess of one orientational isomer over the other in freshly expressed protein results from selective binding of hemin by the apoprotein, a phenomenon that has not heretofore been established for any apocyt b(5).

Exciplex fluorescence in inclusion complexes of naphthalene derivatives

Electronic excitation of compounds 3–5 complexed within the cavity of cyclophane 1 in aqueous solution produces host-guest exciplexes fluorescing at 420 nm. When cyclophane 2 is employed, the complexed guests fluoresce only as monomers. The ratio of exciplex vs. complexed monomer fluorescence is influenced by steric interactions between the host and complexed guest.

Single-Molecule FRET States, Conformational Interchange, and Conformational Selection by Dye Labels in Calmodulin

We investigate the roles of measurement time scale and the nature of the fluorophores in the FRET states measured for calmodulin, a calcium signaling protein known to undergo pronounced conformational changes. The measured FRET distributions depend markedly on the measurement time scale (nanosecond or microsecond). Comparison of FRET distributions measured by donor fluorescence decay with FRET distributions recovered from single-molecule burst measurements binned over time scales of 90 μs to 1 ms reveals conformational averaging over the intervening time regimes. We find further that, particularly in the presence of saturating Ca(2+), the nature of the measured single-molecule FRET distribution depends markedly on the identity of the FRET pair. The results suggest interchange between conformational states on time scales of hundreds of microseconds or less. Interaction with a fluorophore such as the dye Texas Red alters both the nature of the measured FRET distributions and the dynamics of conformational interchange. The results further suggest that the fluorophore may not be merely a benign reporter of protein conformations in FRET studies, but may in fact alter the conformational landscape.

Guest dechlorination and covalent capture following photoexcitation of inclusion complexes in water

Photoexcitation of complexes between cyclophane 1 and 1- or 2-chloronaphthalene in aqueous solution leads to rapid dechlorination of the guest, a reaction driven by electron transfer from host to excited guest. The main photoproducts contain a naphthyl group covalently attached to the host framework. The results may lead to new approaches for remediating water contaminated with chlorinated aromatic compounds.