Conserved C-Terminal Tail Is Responsible for Membrane Localization and Function of Pseudomonas aeruginosa Hemerythrin.

Many bacteria have hemerythrin (Hr) proteins that bind O2, including Pseudomonas aeruginosa, in which microoxia-induced Hr (Mhr) provide fitness advantages under microoxic conditions. Mhr has a 23 amino-acid extension at its C-terminus relative to a well-characterized Hr from Methylococcus capsulatus, and similar extensions are also found in Hrs from other bacteria. The last 11 amino acids of this extended, C-terminal tail are highly conserved in gammaproteobacteria and predicted to form a helix with positively charged and hydrophobic faces. In cellular fractionation assays, wild-type (WT) Mhr was found in both membrane and cytosolic fractions, while a MhrW143* variant lacking the last 11 residues was largely in the cytosol and did not complement Mhr function in competition assays. MhrL112Y, a variant that has a much longer-lived O2-bound form, was fully functional and had a similar localization pattern to that of WT Mhr. Both MhrW143* and MhrL112Y had secondary structures, stabilities, and O2-binding kinetics similar to those of WT Mhr. Fluorescence studies revealed that the C-terminal tail, and particularly the fragment corresponding to its last 11 residues, was sufficient and necessary for association with lipid vesicles. Molecular dynamics simulations and subsequent cellular analysis of Mhr variants have demonstrated that conserved, positively charged residues in the tail are important for Mhr interactions with negatively charged membranes and the contribution of this protein to competitive fitness. Together, these data suggest that peripheral interactions of Mhr with membranes are guided by the C-terminal tail and are independent of O2-binding.

Pseudomonas aeruginosa lasR mutant fitness in microoxia is supported by an Anr-regulated oxygen-binding hemerythrin.

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR-) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR- strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR- strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins.

Multiheme proteins are important in energy conversion and biogeochemical cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model to elucidate roles of the interdomain interface on properties of iron centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with the full-length diheme c4 and its Met-to-His ligand variants, were characterized by a variety of spectroscopic and stability measurements. In both isolated domains, the heme iron is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains, and isolated c4-A is folded. In contrast, the isolated c4-B is partially unfolded, and the interface with c4-A guides folding of this domain. The c4-A and c4-B domains have the propensity to interact even without the polypeptide linker. Thermodynamic cycles have revealed properties of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by the interface. This study illustrates the effects of the interface on tuning structural and redox properties of multiheme proteins and enriches our understanding of redox-dependent complexation.

Comparing Properties of Common Bioinorganic Ligands with Switchable Variants of Cytochrome c.

Ligand substitution at the metal center is common in catalysis and signal transduction of metalloproteins. Understanding the effects of particular ligands, as well as the polypeptide surrounding, is critical for uncovering mechanisms of these biological processes and exploiting them in the design of bioinspired catalysts and molecular devices. A series of switchable K79G/M80X/F82C (X = Met, His, or Lys) variants of cytochrome (cyt) c was employed to directly compare the stability of differently ligated proteins and activation barriers for Met, His, and Lys replacement at the ferric heme iron. Studies of these variants and their nonswitchable counterparts K79G/M80X have revealed stability trends Met < Lys < His and Lys < His < Met for the protein FeIII-X and FeII-X species, respectively. The differences in the hydrogen-bonding interactions in folded proteins and in solvation of unbound X in the unfolded proteins explain these trends. Calculations of free energy of ligand dissociation in small heme model complexes reveal that the ease of the FeIII-X bond breaking increases in the series amine < imidazole < thioether, mirroring trends in hardness of these ligands. Experimental rate constants for X dissociation in differently ligated cyt c variants are consistent with this sequence, but the differences between Met and His dissociation rates are attenuated because the former process is limited by the heme crevice opening. Analyses of activation parameters and comparisons to those for the Lys-to-Met ligand switch in the alkaline transition suggest that ligand dissociation is entropically driven in all the variants and accompanied by Lys protonation at neutral pH. The described thiolate redox-linked switches have offered a wealth of new information about interactions of different protein-derived ligands with the heme iron in cyt c model proteins, and we anticipate that the strategy of employing these switches could benefit studies of other redox metalloproteins and model complexes.

Mechanistic Studies of Proton-Coupled Electron Transfer in a Calorimetry Cell.

Mechanistic studies of proton-coupled electron-transfer (PCET) reactions in proteins are complicated by the challenge of following proton transfer (PT) in these large molecules. Herein we describe the use of isothermal titration calorimetry (ITC) to establish proton involvement in protein redox reactions and the identity of PT sites. We validate this approach with three variants of a heme protein cytochrome c (cyt c) and show that the method yields a wealth of thermodynamic information that is important for characterizing PCET reactions, including reduction potentials, redox-dependent p Ka values, and reaction enthalpies for both electron-transfer (ET) and PT steps. We anticipate that this facile and label-free ITC approach will find widespread applications in studies of other redox proteins and enhance our knowledge of PCET reaction mechanisms.

A Heme Propionate Staples the Structure of Cytochrome c for Methionine Ligation to the Heme Iron.

Ligand-switch reactions at the heme iron are common in biological systems, but their mechanisms and the features of the polypeptide fold that support dual ligation are not well understood. In cytochrome c (cyt c), two low-stability loops (Ω-loop C and Ω-loop D) are connected by the heme propionate HP6. At alkaline pH, the native Met80 ligand from Ω-loop D switches to a Lys residue from the same loop. Deprotonation of an as yet unknown group triggers the alkaline transition. We have created the two cyt c variants T49V/K79G and T78V/K79G with altered connections of these two loops to HP6. Electronic absorption, NMR, and EPR studies demonstrate that at pH 7.4 ferric forms of these variants are Lys-ligated, whereas ferrous forms maintain the native Met80 ligation. Measurements of protein stability, cyclic voltammetry, pH-jump and gated electron-transfer kinetics have revealed that these Thr to Val substitutions greatly affect the alkaline transition in both ferric and ferrous proteins. The substitutions modify the stability of the Met-ligated species and reduction potentials of the heme iron. The kinetics of ligand-switch processes are also altered, and analyses of these effects implicate redox-dependent differences in metal-ligand interactions and the role of the protein dynamics, including cross-talk between the two Ω-loops. With the two destabilized variants, it is possible to map energy levels for the Met- and Lys-ligated species in both ferric and ferrous proteins and assess the role of the protein scaffold in redox-dependent preferences for these two ligands. The estimated shift in the heme iron reduction potential upon deprotonation of the "trigger" group is consistent with those associated with deprotonation of an HP, suggesting that HP6, on its own or as a part of a hydrogen-bonded cluster, is a likely "trigger" for the Met to Lys ligand switch.

The K79G Mutation Reshapes the Heme Crevice and Alters Redox Properties of Cytochrome c.

The two roles of cytochrome c (cyt c), in oxidative phosphorylation and apoptosis, critically depend on redox properties of its heme iron center. The K79G mutant has served as a parent protein for a series of mutants of yeast iso-1 cyt c. The mutation preserves the Met80 coordination to the heme iron, as found in WT* (K72A/C102S), and many spectroscopic properties of K79G and WT* are indistinguishable. The K79G mutation does not alter the global stability, fold, rate of Met80 dissociation, or thermodynamics of the alkaline transition (p Ka) of the protein. However, the reduction potential of the heme iron decreases; further, the p KH of the trigger group and the rate of the Met-to-Lys ligand exchange associated with the alkaline transition decrease, suggesting changes in the environment of the heme. The rates of electron self-exchange and bimolecular electron transfer (ET) with positively charged inorganic complexes increase, as does the intrinsic peroxidase activity. Analysis of the reaction rates suggests that there is increased accessibility of the heme edge in K79G and supports the importance of the Lys79 site for bimolecular ET reactions of cyt c, including those with some of its native redox partners. Structural modeling rationalizes the observed effects to arise from changes in the volume of the heme pocket and solvent accessibility of the heme group. Kinetic and structural analyses of WT* characterize the properties of the heme crevice of this commonly employed reference variant. This study highlights the important role of Lys79 for defining functional redox properties of cyt c.

Ligation and Reactivity of Methionine-Oxidized Cytochrome c.

Met80, one of the heme iron ligands in cytochrome c (cyt c), is readily oxidized to Met sulfoxide (Met-SO) by several biologically relevant oxidants. The modification has been suggested to affect both the electron-transfer (ET) and apoptotic functions of this metalloprotein. The coordination of the heme iron in Met-oxidized cyt c (Met-SO cyt c) is critical for both of these functions but has remained poorly defined. We present electronic absorption, NMR, and EPR spectroscopic investigations as well as kinetic studies and mutational analyses to identify the heme iron ligands in yeast iso-1 Met-SO cyt c. Similar to the alkaline form of native cyt c, Lys73 and Lys79 ligate to the ferric heme iron in the Met80-oxidized protein, but this coordination takes place at much lower pH. The ferrous heme iron is ligated by Met-SO, implying the redox-linked ligand switch in the modified protein. Binding studies with the model peptide microperoxidase-8 provide a rationale for alterations in ligation and for the role of the polypeptide packing in native and Met-SO cyt c. Imidazole binding experiments have revealed that Lys dissociation from the ferric heme in K73A/K79G/M80K (M80K#) and Met-SO is more than 3 orders of magnitude slower than the opening of the heme pocket that limits Met80 replacement in native cyt c. The Lys-to-Met-SO ligand substitution gates ET of ferric Met-SO cyt c with Co(terpy)22+. Owing to the slow Lys dissociation step, ET reaction is slow but possible, which is not the case for nonswitchable M80A and M80K#. Acidic conditions cause Lys replacement by a water ligand in Met-SO cyt c (p Ka = 6.3 ± 0.1), increasing the intrinsic peroxidase activity of the protein. This pH-driven ligand switch may be a mechanism to boost peroxidase function of cyt c specifically in apoptotic cells.

Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome c.

Perturbations in protein structure define the mechanism of allosteric regulation and biological information transfer. In cytochrome c (cyt c), ligation of Met80 to the heme iron is critical for the protein's electron-transfer (ET) function in oxidative phosphorylation and for suppressing its peroxidase activity in apoptosis. The hard base Lys is a better match for the hard ferric iron than the soft base Met is, suggesting the key role of the protein scaffold in favoring Met ligation. To probe the role of the protein structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A were designed to disrupt hydrogen bonding and packing of the heme coordination loop, respectively. Electronic absorption, nuclear magnetic resonance, and electron paramagnetic resonance spectra reveal that ferric forms of both variants are Lys-ligated at neutral pH. A minor change in the tertiary contacts in T49V, away from the heme coordination loop, appears to be sufficient to execute a change in ligation, suggesting a cross-talk between the different regions of the protein structure and a possibility of built-in conformational switches in cyt c. Analyses of thermodynamic stability, kinetics of Lys binding and dissociation, and the pH-dependent changes in ligation provide a detailed characterization of the Lys coordination in these variants and relate these properties to the extent of structural perturbations. The findings emphasize the importance of the hydrogen-bonding network in controlling ligation of the native Met80 to the heme iron.

Redox-dependent stability, protonation, and reactivity of cysteine-bound heme proteins.

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys-heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.