Heme Periphery Research Articles

Electron Transfer from Cytochrome C to Cytochrome C Oxidase: Contribution of Hydrophobic “Breakwater” to Electron Transfer

The electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is the terminal ET reaction in the respiratory chain of mitochondria. By accepting four electrons from the repetitive binding of Cyt c, CcO reduces molecular oxygen to water molecules to promote the proton pumping from the matrix to inner membrane space in mitochondria, resulting in the formation of the proton gradient, which is the primary driving force for the ATP synthesis in the complex V. The ET reaction from Cyt c to CcO is, therefore, the crucial step for the energy generation system in the cellular level. To elucidate regulation mechanisms for the ET reaction from Cyt c to CcO, we determined interaction sites of Cyt c to CcO by NMR measurements and revealed that some hydrophobic amino acid residues including Ile11, Ile81 and Val83 near the exposed heme periphery, in addition to the positively charged amino acid residues as previously reported, are located in the CcO interaction site [1]. Combined with the structural information of the CcO interaction site on Cyt c and the Michaelis-Menten analysis of the steady state kinetics of the ET reaction from Cyt c to CcO, we estimated the structure of the ET complex under turnover conditions using the protein docking simulation. Based on the energetic analysis of the protein-protein interaction in the simulated structure of the ET complex [2], the interactions energetically essential for the complex formation were found to be hydrophobic interactions mediated by several hydrophobic amino acid residues surrounding the exposed heme periphery, which forms a “molecular breakwater”. The formation of such a breakwater would expel hydrated water molecules around the redox centers to form the hydrophobic and effective ET pathway between Cyt c and CcO. The pathway analysis of the ET pathway from Cyt c to CcO in the simulated ET complex between Cyt c and CcO also showed that the ET pathway consists of many hydrophobic amino acid residues in the interaction site and only a few hydrophilic amino acid residues contribute to the ET pathay. To confirm the contribution of the hydrophobic environments to the efficiency of the ET reaction, we mutated one of the hydrophobic amino acid residues constituting the breakwater, Ile81, to a hydrophilic amino acid residue, Ser, and found that the ET rate for the mutant (I81S) was significantly retarded. The simulated structure of the ET complex between I81S Cyt c and CcO also indicated that the reduced stabilization energy from the hydrophobic interactions and disruption of the “breakwater“, due to the invasion of the bulk water to the hydrophobic interaction site between Cyt c and CcO. Less hydrophobic environments of the interaction sites were also supported by the pathway analysis showing more hydrophilic amino acid residues are included in the ET pathway of the I81S mutant complex, compared to that of the wild type complex. However, the products of the decay parameters for the ET pathway, ε tot, reflecting the electronic coupling constant, T DA, for the ET reaction, was increased for the ET complex between I81S Cyt c and CcO, implying that the ET rate should be accelerate by introducing the hydrophilic amino acid residue into the interaction site. On the other hand, recent DFT calculation revealed that direct electron tunneling from the heme moiety of Cyt c to Trp104 near the copper A site of CcO and the lower electron transmission probability, estimated from the Green’s function calculations at the level of DFT, was calculated for the ET reaction in the complex between I81S Cyt c and CcO. Although we have not yet found out the reasons for the discrepancy between the experimental ET rates and the the electronic coupling constant, it is more plausible that electrons would be directly transferred from Cyt c to CcO and formation of such a breakwater would expel hydrated water molecules around the redox centers to form the hydrophobic interaction site to facilitate the effective ET reaction from Cyt c to CcO.

Osmotic pressure effects identify dehydration upon cytochrome c-cytochrome c oxidase complex formation contributing to a specific electron pathway formation.

In the electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO), we determined the number and sites of the hydration water released from the protein surface upon the formation of the ET complex by evaluating the osmotic pressure dependence of kinetics for the ET from Cyt c to CcO. We identified that ∼20 water molecules were dehydrated in complex formation under turnover conditions, and systematic Cyt c mutations in the interaction site for CcO revealed that nearly half of the released hydration water during the complexation were located around Ile81, one of the hydrophobic amino acid residues near the exposed heme periphery of Cyt c. Such a dehydration dominantly compensates for the entropy decrease due to the association of Cyt c with CcO, resulting in the entropy-driven ET reaction. The energetic analysis of the interprotein interactions in the ET complex predicted by the docking simulation suggested the formation of hydrophobic interaction sites surrounding the exposed heme periphery of Cyt c in the Cyt c-CcO interface (a 'molecular breakwater'). Such sites would contribute to the formation of the hydrophobic ET pathway from Cyt c to CcO by blocking water access from the bulk water phase.

Structural Factors for Regulation of Electron Transfer from Cytochrome C to Cytochrome C Oxidase

The electron transfer (ET) reaction from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is the terminal ET reaction in the respiratory chain of mitochondria. By accepting four electrons from the repetitive binding of Cyt c, CcO reduces molecular oxygen to water molecules to promote the proton pumping from the matrix to inner membrane space in mitochondria, resulting in the formation of the proton gradient, which is the primary driving force for the ATP synthesis in the complex V. The ET reaction from Cyt c to CcO is, therefore, the crucial step for the energy generation system in the cellular level. Because the incomplete reduction of molecular oxygen generates reactive oxygen species, which is highly cytotoxic to damage cellular components, the ET reaction from Cyt c to CcO is thought to be strictly regulated and the formation of the specific ET pathway from Cyt c to CcO in the ET complex has been suggested. However, the ET pathway and its regulation mechanism are still controversial due to the lack of the structural information about the ET complex between Cyt c and CcO. To determine the structure of the Cyt c – CcO ET complex, we used the protein-protein docking simulation1 between Cyt c and CcO based on the NMR structural data for the interaction site for CcO on Cyt c 2. The predicted complex structure between Cyt c and CcO clearly showed that the interaction site between the two proteins is highly hydrophobic and the mutational study also revealed that the introduction of hydrophilic amino acid residues significantly reduced the ET rate from Cyt c to CcO3. The formation of such a hydrophobic interaction site was confirmed by the osmotic pressure experiments for the steady-state kinetic measurements of the ET reaction from Cyt c to CcO. Based on the osmotic pressure dependence of Michaelis constant, K m, for the ET reaction from Cyt c to CcO, we found out the significant dehydration from hydrophobic amino acid residues near the exposed hydrophobic heme periphery, facing to the hydrophobic amino acid residues of CcO, to form the hydrophobic interface between Cyt c and CcO in the predicted ET complex. The protein-protein docking simulation for the complex formation with a mutant having the hydrophilic amino acid residues in the interaction site for CcO indicated that a new hydrogen bond was formed in the interaction site between Cyt c and CcO, implying that the incomplete dehydration in the interaction site and/or invasion of the bulk water to the interaction site of the mutant. To determine the ET pathway from Cyt c to CcO, the conventional ET pathway analysis4 was applied to the predicted ET complex between Cyt c and CcO. Although the estimated ET pathway was constructed by many hydrophobic amino acid residues, the introduction of the hydrophobic amino acid residue into the interaction site resulted in the accelerated ET rate in the pathway analysis, which is inconsistent with the experimental data. We re-evaluate the ET pathway using the Green’s function and found that electron is directly transferred from the heme iron of Cyt c to the cupper ion of the subunit II of CcO through the tunneling mechanism, and the decreased hydrophobicity in the interaction site reduced the ET rate in this analysis. We, therefore, conclude that the formation of the hydrophobic interaction site by the dehydration around exposed heme periphery is essential for the regulation of the ET reaction from Cyt c to CcO in the respiratory chain. Sato, W.; Hitaoka, S.; Inoue, K.; Imai, M.; Saio, T.; Uchida, T.; Shinzawa-Itoh, K.; Yoshikawa, S.; Yoshizawa, K.; Ishimori, K., Energetic Mechanism of Cytochrome c-Cytochrome c Oxidase Electron Transfer Complex Formation under Turnover Conditions Revealed by Mutational Effects and Docking Simulation. J. Biol. Chem. 2016, 291 (29), 15320-15331. Sakamoto, K.; Kamiya, M.; Imai, M.; Shinzawa-Itoh, K.; Uchida, T.; Kawano, K.; Yoshikawa, S.; Ishimori, K., NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase. Proc. Natl. Acad. Sci. USA 2011, 108 (30), 12271-12276. Sato, W.; Uchida, T.; Saio, T.; Ishimori, K., Polyethylene glycol promotes autoxidation of cytochrome c. Biochim. Biophys. Acta 2018, 1862 (6), 1339-1349. Onuchic, J. N.; Beratan, D. N.; Winkler, J. R.; Gray, H. B., Pathway Analysis of Protein Electron-Transfer Reactions. Ann. Rev. Biophys. Biomol. Struct. 1992, 21 (1), 349-377.

Structural and Functional Characterization of Electron Transfer Complex Between Cytochrome C and Cytochrome C Oxidase

The electron transfer (ET) reactions in mitochondrial and bacterial respiratory chains are essential processes for energy transduction in cells. In the respiratory chain of mitochondria, the electrons to reduce molecular oxygen at cytochrome c oxidase (CcO) are donated from a small hemoprotein, cytochrome c (Cyt c), and Cyt c is thought to form an ET complex with CcO to promote the ET reaction from the heme iron in Cyt c to the CuA site in CcO. While the reduction of molecular oxygen to water molecules requires four electrons, Cyt c can carry only one electron, implying that Cyt c repetitively associates with and dissociates from CcO and suggesting that the specific interprotein interactions between Cyt c and CcO regulate the binding affinity and the ET rate from Cyt c to CcO. By using NMR spectroscopy, we have successfully determined the interaction site of Cyt c to CcO and found that the hydrophobic interactions are crucial for the complex formation between Cyt c and CcO1. To confirm the energetic contribution of the hydrophobic interaction to the complex formation, we determined the numbers and sites of the dehydrated water molecules associated with formation of the ES (E: CcO, S: reduced Cyt c) complex by the osmotic pressure analysis, presenting the critical contribution of the dehydration to the entropy increase for the ES complex formation. Based on the osmotic pressure dependence of kinetics for the ET from Cyt c to CcO, about 20 water molecules were found to be dehydrated in the complex formation under turnover conditions, and the systematic mutations in the interaction site of Cyt c for CcO revealed that nearly half of the dehydrated water molecules were located around Ile81, one of the hydrophobic amino acid residues near the exposed heme periphery of Cyt c. Such a specific dehydration from the hydrophobic residue, dominantly compensating for the entropy decrease due to the association of Cyt c with CcO, forms a structure blocking water access from the bulk water phase (a “molecular breakwater”) surrounding the ET pathway across the Cyt c – CcO interface in the ES complex to protect the ET pathway from the leak of electrons to the bulk water phase. To get further insights into the ET pathway from Cyt c to CcO, we determined the structure of the electron transfer complex between Cyt c and CcO under turnover conditions by considering the mutational effects on the steady state kinetics of the electron transfer reaction and our NMR analysis of the interaction site, and energetically characterized the interactions essential for complex formation2. The complex structures predicted by the protein docking simulation were computationally selected and validated by the experimental kinetic data for mutant Cyt c in the electron transfer reaction to CcO. The interaction analysis using the selected Cyt c – CcO complex structure revealed the electrostatic and hydrophobic contributions of each amino acid residue to the free energy required for complex formation. Several charged residues showed large unfavorable (desolvation) electrostatic interactions which were almost cancelled out by large favorable (Columbic) electrostatic interactions, but resulting in the destabilization of the complex. The residual destabilizing free energy is compensated by the van der Waals interactions mediated by hydrophobic amino acid residues to give the stabilized complex. Thus, hydrophobic interactions are the primary factors that promote complex formation between Cyt c and CcO under turnover conditions, while the change in the electrostatic destabilization free energy provides the variance of the binding free energy in the mutants. The distribution of favorable and unfavorable electrostatic interactions in the interaction site determines the orientation of the binding of Cyt c on CcO. Based on the simulated complex structure between Cyt c and CcO, we examined the contribution of hydrophobic interactions to the ET rate. The replacement of Ile81 in Cyt c, a key hydrophobic amino acid residue constituting the hydrophobic region in the protein interface between Cyt c and CcO in the ET complex, with Ser reduced the ET rate and the Marcus theory suggested that the significant decrease of electronic coupling constant (H DA), corresponding to the perturbations in the ET pathway from Cyt c to CcO. The dehydration of Ile81, therefore, facilitates the ET reaction from Cyt c to CcO by constituting the interprotein hydrophobic region as well as entropically promotes the ET complex formation. 1. Sakamoto, K. et al., Proc. Natl. Acad. Sci. USA 108, 12271-12276, (2011). 2. Sato, W. et al., J. Biol. Chem. 291, 15320-15331, (2016).

Novel bis-arylalkylamines as myeloperoxidase inhibitors: Design, synthesis, and structure-activity relationship study

Human myeloperoxidase (MPO) plays an important role in innate immunity but also aggravates tissue damage by oxidation of biomolecules at sites of inflammation. As a result from a recent high-throughput virtual screening approach for MPO inhibitors, bis-2,2′-[(dihydro-1,3(2H,4H) pyrimidinediyl)bis(methylene)]phenol was detected as a promising lead compound for inhibition of the MPO-typical two-electron oxidation of chloride to hypochlorous acid (IC50 = 0.5 μM). In the present pharmacomodulation study, 37 derivatives of this lead compound were designed and synthesized driven by comprehensive docking studies and the impact on the chlorination activity of MPO. We describe the structural requirements for optimum (i) binding to the heme periphery and (ii) inhibition capacity. Finally, the best three inhibitors (bis-arylalkylamine derivatives) were probed for interaction with the MPO redox intermediates Compound I and Compound II. Determined apparent bimolecular rate constants together with determination of reduction potential and nucleophilicity of the selected compounds allowed us to propose a mechanism of inhibition. The best inhibitor was found to promote the accumulation of inactive form of MPO-Compound II and has IC50 = 54 nM, demonstrating the successful approach of the drug design. Due to the similarity of ligand interactions between MPO and serotonine transporter, the selectivity of this inhibitor was also tested on the serotonin transporter providing a selectivity index of 14 (KiSERT/IC50MPO).

Utility of heme analogues to intentionally modify heme–globin interactions in myoglobin

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme–globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized.Proton NMR spectroscopy revealed that loose heme–globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron–histidine bond. The full rotational rate was estimated to be about 1400s−1 at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme–globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005mmHg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin.Heme–globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics – the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity.

Experimental Documentation of the Structural Consequences of Hydrogen‐Bonding Interactions to the Proximal Cysteine of a Cytochrome P450

Resonance Raman spectroscopy is used to document, for the first time, a 6 cm−1 decrease of the Fe-S stretch by introducing an H-bond donor into the proximal pocket of a cytochrome P450, which interacts with the key cysteine thiolate axial ligand. The anticipated trans-effect on bound exogenous ligands is also confirmed and evidence is obtained supporting intimate interaction of the new histidyl-imidazole fragment with the heme periphery.

NMR basis for interprotein electron transfer gating between cytochrome c and cytochrome c oxidase

The final interprotein electron transfer (ET) in the mammalian respiratory chain, from cytochrome c (Cyt c) to cytochrome c oxidase (CcO) is investigated by (1)H-(15)N heteronuclear single quantum coherence spectral analysis. The chemical shift perturbation in isotope-labeled Cyt c induced by addition of unlabeled CcO indicates that the hydrophobic heme periphery and adjacent hydrophobic amino acid residues of Cyt c dominantly contribute to the complex formation, whereas charged residues near the hydrophobic core refine the orientation of Cyt c to provide well controlled ET. Upon oxidation of Cyt c, the specific line broadening of N-H signals disappeared and high field (1)H chemical shifts of the N-terminal helix were observed, suggesting that the interactions of the N-terminal helix with CcO are reduced by steric constraint in oxidized Cyt c, while the chemical shift perturbations in the C-terminal helix indicate notable interactions of oxidized Cyt c with CcO. These results suggest that the overall affinity of oxidized Cyt c for CcO is significantly, but not very much weaker than that of reduced Cyt c. Thus, electron transfer is gated by dissociation of oxidized Cyt c from CcO, the rate of which is controlled by the affinity of oxidized Cyt c to CcO for providing an appropriate electron transfer rate for the most effective energy coupling. The conformational changes in Lys13 upon CcO binding to oxidized Cyt c, shown by (1)H- and (1)H, (15)N-chemical shifts, are also expected to gate intraprotein ET by a polarity control of heme c environment.

Sites of Covalent Attachment of CYP4 Enzymes to Heme: Evidence for Microheterogeneity of P450 Heme Orientation

Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link furnished, in part, by a conserved I-helix acid, Glu310. Chromatography, mass spectrometry, and NMR have now been utilized to identify the site of attachment on the heme. Native CYP4B1 covalently binds heme solely at the C-5 methyl position. Unexpectedly, recombinant CYP4B1 from insect cells and Escherichia coli also bound their heme covalently at the C-8 methyl position. Structural heterogeneity may be common among recombinant CYP4 proteins because CYP4A3 exhibited this duality. Attempts to evaluate functional heterogeneity were complicated by the complexity of the system. The phenomenon of covalent heme binding to P450 provides a novel method for assessing microheterogeneity in heme orientation and raises questions about the fidelity of heme incorporation in recombinant systems.

Reversible binding of nitric oxide and carbon-carbon bond formation in a meso-hydroxylated heme.

Exposure of a pyridine solution of (py)2Fe(OEPO) (1) (OEPO is the trianion of octaethyloxophlorin, py is pyridine) at 22 degrees C to nitric oxide under the strict exclusion of dioxygen results in a color change from green to red-brown and the formation of {(py)(ON)Fe(OEPO)}2 (2). Monitoring the reaction by 1H NMR spectroscopy shows that the reaction is reversible. The air-sensitive product {(py)(ON)Fe(OEPO)}2 has been isolated and characterized by X-ray crystallography. NO binding is accompanied by association of two hemes through the formation of a new C-C bond between two meso carbon atoms on the porphyrin periphery. The macrocyclic ligand has a ruffled distortion that positions the C5-O1 group with its short C=O bond distance (1.237(3) A) in close proximity to the NO ligand. Since further attack upon 1 by O2 during heme degradation involves reaction in the vicinity of the oxygenated meso position, the positioning of the NO ligand so that the O2...C5 distance is only 3.100(3) A presents a highly suggestive model for the next stage of attack upon the heme periphery.

Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450.

We elucidate the hydroxylation of camphor by cytochrome P450 with the use of density functional and mixed quantum mechanics/molecular mechanics methods. Our results reveal that the enzyme catalyzes the hydrogen-atom abstraction step with a remarkably low free-energy barrier. This result provides a satisfactory explanation for the experimental failure to trap the proposed catalytically competent high-valent heme Fe(IV) oxo (oxyferryl) species responsible for this hydroxylation chemistry. The primary and previously unappreciated contribution to stabilization of the transition state is the interaction of positively charged residues in the active-site cavity with carboxylate groups on the heme periphery. A similar stabilization found in dioxygen binding to hemerythrin, albeit with reversed polarity, suggests that this mechanism for controlling the relative energetics of redox-active intermediates and transition states in metalloproteins may be widespread in nature.

Resonance Raman evidence for protein-induced out-of-plane distortion of the heme prosthetic group of mammalian lactoperoxidase.

Resonance Raman spectra have been acquired for resting state mammalian lactoperoxidase, LPO(N), and its six-coordinate, low-spin (6CLS) cyanide complex, LPO(CN), as well as for various heme l containing fragments resulting from partial or complete proteolytic digestion. These proteolytic fragments provide a useful set of reference compounds for analysis of the LPO(N) and LPO(CN) enzymes, using various ligands to generate well-defined five-coordinate and six-coordinate high-spin (5CHS and 6CHS) species. In addition, these model compounds, which contain zero, one, or two covalently attached ester linkages to polypeptide chains, are quite useful for determining the extent to which the presence of the ester linkages at the heme periphery affects the characteristic heme resonance Raman marker bands. The spectral results not only provide strong evidence for the formulation of the resting state enzyme as a 6CHS species, but also confirm the previously documented anomalous intensities of several low-frequency resonance Raman bands, which are most reasonably interpreted to arise from a protein-induced out-of-plane distortion of the heme l macrocycle mediated by the covalent ester linkages to the associated polypeptide residues of the intact protein.

Properties and reactivity of myoglobin reconstituted with chemically modified protohemin complexes.

The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.

Q-Band resonance Raman investigation of turnip cytochrome f and Rhodobacter capsulatus cytochrome c1

The results of a comprehensive Q-band resonance Raman investigation of cytochrome c 1 and cytochrome f subunits of bc 1 and b 6 f complexes are presented. Q-band excitation provides a particularly effective probe of the local heme environments of these species. The effects of protein conformation (particularly axial ligation) on heme structure and function were further investigated by comparison of spectra obtained from native subunits to those of a site directed c 1 mutant (M183L) and various pH-dependent species of horse heart cytochrome c. In general, all species examined displayed variability in their axial amino acid ligation that suggests a good deal of flexibility in their hemepocket conformations. Surprisingly, the large scale protein rearrangements that accompany axial ligand replacement have little or no effect on macrocycle geometry in these species. This indicates the identity and/or conformation of the peptide linkage between the two cysteines that are covalently linked to the heme periphery may determine heme geometry.

Solution 1H NMR investigation of the heme cavity and substrate binding site in cyanide-inhibited horseradish peroxidase.

Solution two-dimensional 1H NMR studies have been carried out on cyanide-inhibited horseradish peroxidase isozyme C (HRPC-CN) to explore the scope and limitations of identifying residues in the heme pocket and substrate binding site, including those of the "second sphere" of the heme, i.e. residues which do not necessarily have dipolar contact with the heme. The experimental methods use a range of experimental conditions to obtain data on residue protons with a wide range of paramagnetic relaxivity. The signal assignment strategy is guided by the recently reported crystal structure of recombinant HRPC and the use of calculated magnetic axes. The goal of the assignment strategy is to identify signals from all residues in the heme, as well as proximal and distal, environment and the benzhydroxamic acid (BHA) substrate binding pocket. The detection and sequence specific assignment of aromatic and aliphatic residues in the vicinity of the heme pocket confirm the validity of the NMR methodologies described herein. Nearly all residues in the heme periphery are now assigned, and the first assignments of several "second sphere" residues in the heme periphery are reported. The results show that nearly all catalytically relevant amino acids in the active site can be identified by the NMR strategy. The residue assignment strategy is then extended to the BHA:HRPC-CN complex. Two Phe rings (Phe 68 and Phe 179) and an Ala (Ala 140) are shown to be in primary dipolar contact to BHA. The shift changes induced by substrate binding are shown to reflect primarily changes in the FeCN tilt from the heme normal. The present results demonstrate the practicality of detailed solution 1H NMR investigation of the manner in which substrate binding is perturbed by either variable substrates or point mutations of HRP.

Evidence for differential binding of isoniazid by Mycobacterium tuberculosis KatG and the isoniazid-resistant mutant KatG(S315T).

Isoniazid is a mainstay of antibiotic therapy for the treatment of tuberculosis, but its molecular mechanism of action is unclear. Previous investigators have hypothesized that isoniazid is a prodrug that requires in vivo activation by KatG, the catalase-peroxidase of Mycobacterium tuberculosis, and that resistance to isoniazid strongly correlates with deletions or point mutations in KatG. One such mutation, KatG(S315T), is found in approximately 50% of clinical isolates exhibiting isoniazid resistance. In this work, 1H nuclear magnetic resonance T1 relaxation measurements indicate that KatG and KatG(S315T) each bind isoniazid at a position approximately 12 A from the active site heme iron. Electron paramagnetic resonance spectroscopy revealed heterogeneous populations of high-spin ferric heme in both wild-type KatG and KatG(S315T) with the ratios of each species differing between the two enzymes. Small changes in the proportions of these high-spin species upon addition of isoniazid support the finding that isoniazid binds near the heme periphery of both enzymes. Titration of wild-type KatG with isoniazid resulted in the appearance of a "type I" substrate-induced difference spectrum analogous to those seen upon substrate binding to the cytochromes P450. The difference spectrum may result from an isoniazid-induced change in a portion of the KatG heme iron from 6- to 5-coordinate. Titration of KatG(S315T) with isoniazid failed to produce a measurable difference spectrum indicating an altered active site configuration. These results suggest that KatG(S315T) confers resistance to isoniazid through subtle changes in the isoniazid binding site.

Control of Myoglobin Electron-Transfer Rates by the Distal (Nonbound) Histidine Residue

Changing the distal histidine (H64) of sperm-whale myoglobin into any of the following residuesvaline, leucine, methionine, glycine, or phenylalaninecauses a dramatic improvement in the reversibility (electron-transfer kinetics) and reproducibility of the direct electrochemistry. Cyclic voltammograms of native or wild-type recombinant myoglobin are irreversible (in the electrochemical sense) and critically dependent on the condition of the sample and electrode. By contrast, the H64 mutants display quasi-reversible electrochemistry much more typical of results obtained with true electron-transfer proteins. The difference in activity correlates sharply with alterations to the distal-pocket H-bonding network, which in the native protein comprises the H2O that is coordinated to Fe(III), the Nε of H64, and the “lattice” extending from arginine-45 to the heme periphery. It is proposed that this H-bond network increases the electron-transfer activation energy by coupling the displacement of Fe(III)-coordinated H2O to higher reorganization requirements, including that of solvent H2O molecules near the heme periphery. The poor reproducibility and extreme sensitivity of the electrochemical response to experimental conditions is rationalized by the microscopic model for protein electrochemistry which predicts that the waveshape and potential positions for inherently irreversible (sluggish) systems will be critically dependent on the state of the electrode surface.

Horseradish peroxidase inhibition by thiouracils

In this paper, the activity of horseradish peroxidase was further determined in the presence of several uracil derivatives. The rate of guaiacol peroxidation decreases in presence of 2-thiouracil and of 6- n-propyl-2-thiouracil, but is not changed by 6- n-propyluracil nor uracil. Thus, thiouracils inhibit horseradish peroxidase in a noncompetitive form. The binding of 6- n-propyl-2-thiouracil, 2-thiouracil, 6- n-propyluracil and uracil with horseradish peroxidase shows difference spectra due to changes in the environment of heme group in peroxidase. Then, the binding sites for these uracil derivatives are in an hydrophobic pocket at the heme periphery of peroxidase. The lesser binding rates were for uracil and propyluracil, which did not inhibit the peroxidase activity. These results point to the thiol group in uracils as responsible for the inhibition of peroxidase activity through interaction with an allosteric binding site, in peroxidase heme environment.

Proton NMR study of the heme complex of hemopexin

Proton nuclear magnetic resonance spectroscopy of the complex of heme with hemopexin, a plasma protein with an exceptionally high affinity for heme, is reported. Characteristic spectra are shown for heme · hemopexin of cow, human, rabbit and rat. Each of these spectra demonstrate that the iron of heme bound by hemopexin is paramagnetic and low-spin. Rabbit heme · hemopexin, which exhibits the best signal-to-noise ratio, is studied in detail. Deuterium isotope labelling experiments indicate that the methyl in heme positions 1-, 3- and 8- are resolved downfield from the protein envelope of resonances; the 5-methyl may llie in the −5 to +12 ppm region. Two-dimensional nuclear Overhauser effect locates other protons of the heme periphery, including from the 2-vinyl. Strongly relaxed upfield resonances are identified and assigned to protons on the axial ligands. Cyanide interaction with heme · hemopexin produces an additional low-spin adduct.