Kenton R. Rodgers

Heme-based sensors in biological systems.

The past several years have been witness to a staggering rate of advancement in the understanding of how organisms respond to changes in the availability of diatomic molecules that are toxic and/or crucial to survival. Heme-based sensors presently constitute the majority of the proteins known to sense NO, O2 and CO and to initiate the chemistry required to adapt to changes in their availabilities. Knowledge of the three characterized members of this class, soluble guanylate cyclase, FixL and CooA, has grown substantially during the past year. The major advances have resulted from a broad range of approaches to elucidation of both function and mechanism. They include growth in the understanding of the interplay between the heme and protein in soluble guanylate cyclase, as well as alternate means for its stimulation. Insight into the O2-induced structural changes in FixL has been supplied by the single crystal structure of the heme domain of Bradyrhizobium japonicum. Finally, the ligation environment and ligand interchange that facilitates CO sensing by CooA has been established by spectroscopic and mutagenesis techniques.

Is the Bis(μ‐oxo)dicopper Core Capable of Hydroxylating an Arene?

Direct attack of the bis(μ-oxo)dicopper core on an arene appears feasible in tyrosinase and model complexes on the basis of studies of new [Cu(III) 2 (μ-O)2 ](2+) compounds supported by bidentate imine/amine ligands. In the first demonstration of such reactivity for a bis(μ-oxo)dicopper core, decomposition of these intermediates caused hydroxylation of a pendant phenyl ring [Eq. (a)] in a reaction analogous to that catalyzed by tyrosinase.

The heme transfer from the soluble HasA hemophore to its membrane-bound receptor HasR is driven by protein-protein interaction from a high to a lower affinity binding site.

HasA is an extracellular heme binding protein, and HasR is an outer membrane receptor protein from Serratia marcescens. They are the initial partners of a heme internalization system allowing S. marcescens to scavenge heme at very low concentrations due to the very high affinity of HasA for heme (Ka = 5,3 × 1010 m-1). Heme is then transferred to HasR, which has a lower affinity for heme. The mechanism of the heme transfer between HasA and HasR is largely unknown. HasR has been overexpressed and purified in holo and apo forms. It binds one heme molecule with a Ka of 5 × 106 m-1 and shows the characteristic absorbance spectrum of a low spin heme iron. Both holoHasA and apoHasA bind tightly to apoHasR in a 1:1 stoichiometry. In this study we show that heme transfer occurs in vitro in the purified HasA·HasR complex, demonstrating that heme transfer is energy- and TonB complex-independent and driven by a protein-protein interaction. We also show that heme binding to HasR involves two conserved histidine residues.

The Cytoplasmic Heme-binding Protein (PhuS) from the Heme Uptake System of Pseudomonas aeruginosa Is an Intracellular Heme-trafficking Protein to the δ-Regioselective Heme Oxygenase

The uptake and utilization of heme as an iron source is a receptor-mediated process in bacterial pathogens and involves a number of proteins required for internalization and degradation of heme. In the following report we provide the first in-depth spectroscopic and functional characterization of a cytoplasmic heme-binding protein PhuS from the opportunistic pathogen Pseudomonas aeruginosa. Spectroscopic characterization of the heme-PhuS complex at neutral pH indicates that the heme is predominantly six-coordinate low spin. However, the resonance Raman spectra and global fit analysis of the UV-visible spectra show that at all pH values between 6 and 10 three distinct species are present to varying degrees. The distribution of the heme across multiple spin states and coordination number highlights the flexibility of the heme environment. We provide further evidence that the cytoplasmic heme-binding proteins, contrary to previous reports, are not heme oxygenases. The degradation of the heme-PhuS complex in the presence of a reducing agent is a result of H2O2 formed by direct reduction of molecular oxygen and does not yield biliverdin. In contrast, the heme-PhuS complex is an intracellular heme trafficking protein that specifically transfers heme to the previously characterized iron-regulated heme oxygenase pa-HO. Surface plasmon resonance experiments confirm that the transfer of heme is driven by a specific protein-protein interaction. This data taken together with the spectroscopic characterization is consistent with a protein that functions to shuttle heme within the cell.

How Active-Site Protonation State Influences the Reactivity and Ligation of the Heme in Chlorite Dismutase

Chlorite dismutase catalyzes O(2) release from chlorite with exquisite efficiency and specificity. The spectroscopic properties, ligand binding affinities, and steady-state kinetics of chlorite dismutase from Dechloromonas aromatica were examined over pH 3-11.5 to gain insight into how the protonation state of the heme environment influences dioxygen formation. An acid-base transition was observed by UV/visible and resonance Raman (rR) spectroscopy with a pK(a) of 8.7, 2-3 pH units below analogous transitions observed in typical His-ligated peroxidases. This transition marks the conversion of a five-coordinate high-spin Fe(III) to a mixed high/low-spin ferric hydroxide, as confirmed by rR spectroscopy. The two Fe-OH stretching frequencies are quite low, consistent with a weak Fe-OH bond, despite the nearly neutral imidazole side chain of the proximal histidine ligand. The hydroxide is proposed to interact strongly with a distal H-bond donor, thereby weakening the Fe-OH bond. The rR spectra of Cld-CO as a function of pH reveal two forms of the complex, one in which there is minimal interaction of distal residues with the carbonyl oxygen and another, acidic form in which the oxygen is under the influence of positive charge. Recent crystallographic data reveal arginine 183 as the lone H-bond-donating residue in the distal pocket. It is likely that this Arg is the strong, positively charged H-bond donor implicated by vibrational data to interact with exogenous axial heme ligands. The same Arg in its neutral (pK(a) approximately 6.5) form also appears to act as the active-site base in binding reactions of protonated ligands, such as HCN, to ferric Cld. The steady-state profile for the rate of chlorite decomposition is characterized by these same pK(a) values. The five-coordinate high-spin acidic Cld is more active than the alkaline hydroxide-bound form. The acid form decomposes chlorite most efficiently when the distal Arg is protonated/cationic (maximum k(cat) = 2.0(+/-0.6) x 10(5) s(-1), k(cat)/K(M) = 3.2(+/-0.4) x 10(7) M(-1) s(-1), pH 5.2, 4 degrees C) and to a somewhat lesser extent when it acts as a H-bond donor to the axial hydroxide ligand under alkaline conditions.

Cobalt-Dinitrogen Complexes with Weakened N-N Bonds

Reported N(2) complexes of cobalt do not have substantial weakening of the N-N bond. Using diketiminate ligands to enforce three-coordinate geometries, we have synthesized several novel CoNNCo complexes. In formally univalent complexes, cobalt is poorer than iron at weakening the N-N bond, but in formally zerovalent complexes, cobalt and iron give similar N-N weakening. The weakening is due to cobalt-to-N(2) pi-backbonding, and potassium cations pull more electron density into N(2). These results show that the low coordination number of a trigonal-planar geometry is impetus enough to make even the electronegative cobalt weaken the N-N bond of N(2).

Heme-protein interactions in cytochrome c peroxidase revealed by site-directed mutagenesis and resonance Raman spectra of isotopically labeled hemes

Isotope labeling has been used to assign the resonance Raman spectra of cytochrome c peroxidase, expressed in Escherichia coli [CCP (MKT)], and of the D235N site mutant. 54Fe labeling establishes the coexistence of two separate bands (233 and 246 cm-1), arising from the stretching of the bond between the Fe atom and the proximal histidine ligand, His175. These are assigned to tautomers of the H-bond between the His175 imidazole NΓH proton and the Asp235 carboxylate side chain: In one tautomer the proton resides on the imidazole while in the other the proton is transferred to the carboxylate. When Asp235 is replaced by Asn, the H-bond is lost, and the Fe-His stretching frequency is markedly lowered. Two new RR bands are produced, at 205 and 185 cm-1, as a result of coupling between the shifted Fe-His vibration and a nearby porphyrin mode; the two bands share the 54Fe sensitivity expected for Fe-His stretching. C=C stretching and CβC=C bending vibrations have been separately assigned to the 2- and 4-vinyl groups of the protoheme prosthetic group via selective vinyl deuteration. In the acid form of the enzyme, the frequencies coincide for the two vinyl groups, at 1618 cm-1 for the C=C stretch, and at 406 cm-1 for the CβC=C bend. However, the 2-vinyl frequencies are elevated in the alkaline form of the enzyme, to 1628 cm-1 for C=C stretching, and to 418 cm-1 for CβC=C bending, while the 4-vinyl frequencies remain unshifted. Thus, the acid-alkaline transition involves a protein conformation change that specifically perturbs the 2-vinyl substituent. This perturbation might be a reorientation of the vinyl group, or an alteration of the porphyrin geometry that affects the porphyrin-vinyl coupling. The perturbation is attenuated when CO is bound to the enzyme; the C=C frequency is then unaffected in the alkaline form, while the CβC=C bending frequency is shifted to a smaller extent (412 cm-1). This attenuation is probably linked to inhibition of distal histidine binding to the heme Fe in the alkaline form when the CO is bound.

Deciphering the structural role of histidine 83 for heme binding in hemophore HasA.

Heme carrier HasA has a unique type of histidine/tyrosine heme iron ligation in which the iron ion is in a thermally driven two spin states equilibrium. We recently suggested that the H-bonding between Tyr75 and the invariantly conserved residue His83 modulates the strength of the iron-Tyr75 bond. To unravel the role of His83, we characterize the iron ligation and the electronic properties of both wild type and H83A mutant by a variety of spectroscopic techniques. Although His83 in wild type modulates the strength of the Tyr-iron bond, its removal causes detachment of the tyrosine ligand, thus giving rise to a series of pH-dependent equilibria among species with different axial ligation. The five coordinated species detected at physiological pH may represent a possible intermediate of the heme transfer mechanism to the receptor.

Ist der Bis(μ‐oxo)dikupfer‐Kern fähig, ein Aren zu hydroxylieren?

Der direkte Angriff des Bis(μ-oxo)dikupfer-Kerns auf ein Aren scheint auf der Grundlage von Studien an neuen [CuIII2(μ-O)2]2 +-Verbindungen, die durch zweizahnige Imin/Amin-Liganden stabilisiert werden, in Tyrosinase und Modellkomplexen moglich zu sein. Anhand der Reaktivitat eines Bis(μ-oxo)dikupfer-Kerns wird gezeigt, das der Zerfall dieses Intermediates einen gebundenen Phenylring in einer Reaktion, die der durch Tyrosinase katalysierten analog ist, hydroxyliert [Gl. (a)].

Heme-based Sensing by the Mammalian Circadian Protein, CLOCK

Heme is emerging as a key player in the synchrony of circadian-coupled transcriptional regulation. Current evidence suggests that levels of circadian-linked transcription are regulated in response to both the availability of intracellular heme and heme-based sensing of carbon monoxide (CO) and possibly nitric oxide (NO). The protein CLOCK is central to the regulation and maintenance of circadian rhythms in mammals. CLOCK comprises two PAS domains, each with a heme binding site. Our studies focus on the functionality of the murine CLOCK PAS-A domain (residues 103-265). We show that CLOCK PAS-A binds iron(III) protoporhyrin IX to form a complex with 1:1 stoichiometry. Optical absorbance and resonance Raman studies reveal that the heme of ferric CLOCK PAS-A is a six-coordinate, low-spin complex whose resonance Raman signature is insensitive to pH over the range of protein stability. Ferrous CLOCK PAS-A is a mixture of five-coordinate, high-spin and six-coordinate, low-spin complexes. Ferrous CLOCK PAS-A forms complexes with CO and NO. Ferric CLOCK PAS-A undergoes reductive nitrosylation in the presence of NO to generate a CLOCK PAS-A-NO, which is a five-coordinate {FeNO}(7) complex. Formation of the highly stable {FeNO}(7) heme complex from either ferrous or ferric heme makes possible the binding of NO at very low concentration, a characteristic of NO sensors. Comparison of the spectroscopic properties and CO-binding kinetics of CLOCK PAS-A with other CO sensor proteins reveals that CLOCK PAS-A exhibits chemical properties consistent with a heme-based gas sensor protein.