Proximal His Research Articles

Electronic Structure of Heme-Nitrosyls and Its Significance for Nitric Oxide Reactivity, Sensing, Transport, and Toxicity in Biological Systems

This review summarizes recent developments in the investigation of the electronic structures, spectroscopic properties, and reactivities of ferrous and ferric heme-nitrosyls and how this relates to important biological processes. Ferrous heme-nitrosyls show interesting variations in electronic structure as a function of the different types of proximal ligands, as is evident from electron paramagnetic resonance, magnetic circular dichroism, and vibrational spectroscopy. In particular, coordination of imidazoles like histidine (His) increases the radical character on NO and, in this way, could help activate the bound NO for catalysis. Vice versa, the bound NO ligand imposes a strong sigma trans effect on the proximal His, which, in the case of soluble guanylate cyclase (sGC), the biological NO sensor protein, induces breaking of the Fe(II)-His bond and activates the protein. The possibility of sGC activation by HNO is also discussed. Finally, the properties of ferrous heme-nitrosyls with proximal cysteinate (Cys) coordination are evaluated. It has been known for some time that ferric heme-nitrosyls are intrinsically more labile than their ferrous counterparts, but the underlying reasons for this observation have not been clarified. New results show that this property relates to the presence of a low-lying excited state that is dissociative with respect to the Fe(III)-NO bond. On the other hand, the ground state of these complexes is best described as Fe(II)-NO(+), which shows a very strong Fe-NO bond, as is evident from vibrational spectroscopy. NO, therefore, is a weak ligand to ferric heme, which, at the same time, forms a strong Fe-NO bond. This is possible because the thermodynamic weakness and spectroscopic strength of the Fe-NO bond relate to the properties of different electronic states. Thiolate coordination to ferric hemes leads to a weakening of both the Fe-NO and N-O bonds as a function of the thiolate donor strength. This observation can be explained by a sigma backbond into the sigma* orbital of the Fe-N-O unit that is mediated by the thiolate sigma-donor orbital via orbital mixing. This is a new interaction in heme-nitrosyl that has not been observed before. This also induces a bending of the Fe-N-O subunit in these cases. New spectroscopic data on a corresponding model complex are included in this paper. Finally, the mechanism of NO reduction by cytochrome P450nor is elucidated based on recent density functional theory results.

Unusual fascicular extrasystoles: where is the site of origin?

Radiofrequency ablation for symptomatic, drug refractory premature ventricular contractions has been well established as a treatment modality. Mapping of ventricular ectopy that arises near the native conduction system can be challenging. We report a case of fascicular ectopy arising from the proximal His (H)-Purkinje system that was successfully mapped and ablated. The use of surface and intracardiac electrograms and timing of the His bundle and/or Purkinje potentials is crucial in identifying the exact site of origin.

EPR and Mössbauer Spectroscopy Show Inequivalent Hemes in Tryptophan Dioxygenase

Tryptophan 2,3-dioxygenase (TDO) is an essential enzyme in the pathway of NAD biosynthesis and important for all living organisms. TDO catalyzes oxidative cleavage of the indole ring of L-tryptophan (L-Trp), converting it to N-formylkynurenine (NFK). The crystal structure of TDO shows a dimer of dimer quaternary structure of the homotetrameric protein. The four catalytic sites of the protein, one per subunit, contain a heme that catalyzes the activation and insertion of dioxygen into L-Trp. Because of the alpha(4) structure and because only one type of heme center has been identified in previous spectroscopic studies, the four hemes sites have been presumed to be equivalent. The present work demonstrates that the heme sites of TDO are not equivalent. Quantitative interpretation of EPR and Mössbauer spectroscopic data indicates the presence of two dominant inequivalent heme species in reduced and oxidized states of the enzyme, which is consistent with a dimer of dimer protein quaternary structure that now extends to the electronic properties of the hemes. The electronic properties of the hemes in the reduced state of TDO change significantly upon L-Trp addition, which is attributed to a change in the protonation state of the proximal histidine to the hemes. The binding of O(2) surrogates NO or CO shows two inequivalent heme sites. The heme-NO complexes are 5- and 6-coordinate without L-Trp, and both 6-coordinate with L-Trp. NO can be selectively photodissociated from only one of the heme-NO sites and only in the presence of L-Trp. Cryoreduction of TDO produces a novel diamagnetic heme species, tentatively assigned as a reduced heme-OH complex. This work presents a new description of the heme interactions with the protein, and with the proximal His, which must be considered during the general interpretation of physical data as it relates to kinetics, mechanism, and function of TDO.

Paramagnetic 13C and 15N NMR Analyses of the Push and Pull Effects in Cytochrome c Peroxidase and Coprinus cinereus Peroxidase Variants: Functional Roles of Highly Conserved Amino Acids around Heme

Paramagnetic (13)C and (15)N nuclear magnetic resonance (NMR) spectroscopy of heme-bound cyanide ((13)C(15)N) was applied to 11 cytochrome c peroxidase (CcP) and Coprinus cinereus peroxidase (CIP) mutants to investigate contributions to the push and pull effects of conserved amino acids around heme. The (13)C and (15)N NMR data for the distal His and Arg mutants indicated that distal His is the key amino acid residue creating the strong pull effect and that distal Arg assists. The mutation of distal Trp of CcP to Phe, the amino acid at this position in CIP, changed the push and pull effects so they resembled those of CIP, whereas the mutation of distal Phe of CIP to Trp changed this mutant to become CcP-like. The (13)C NMR shifts for the proximal Asp mutants clearly showed that the proximal Asp-His hydrogen bonding strengthens the push effect. However, even in the absence of a hydrogen bond, the push effect of proximal His in peroxidase is significantly stronger than in globins. Comparison of these NMR data with the compound I formation rate constants and crystal structures of these mutants showed that (1) the base catalysis of the distal His is more critical for rapid compound I formation than its acid catalysis, (2) the primary function of the distal Arg is to maintain the distal heme pocket in favor of rapid compound I formation via hydrogen bonding, and (3) the push effect is the major contributor to the differential rates of compound I formation in wild-type peroxidases.

Ligand orientation of human neuroglobin obtained from solution NMR and molecular dynamics simulation as compared with X-ray crystallography

Neuroglobin, a new member of hemoprotein family, can reversibly bind oxygen and take part in many biological processes such as enzymatic reaction, signal transduction and the mitochondria function. Different from myoglobin and hemoglobin, it has a hexacoordinated heme environment, with histidyl imidazole of proximal His(96)(F8) and distal His(64)(E7) directly bound to the metal ion. In the present work, solution (1)H NMR spectroscopy was employed to investigate the electronic structure of heme center of wild-type met-human neuroglobin. The resonances of heme protons and key residues in the heme pocket were assigned. Two heme orientations resulting from a 180 degrees rotation about the alpha-gamma-meso axis with a population ratio about 2:1 were observed. Then the (1)H NMR chemical shifts of the ferriheme methyl groups were used to predict orientations of the axial ligand. The obtained axial ligand plane angle phi is consistent with that from the molecular dynamics simulation but not with those from the crystal data. Compared with mouse neuroglobin, the obtained average ligand orientation of human neuroglobin reflects the changeability of heme environment for the Ngb family.

Surface Modification Affects the Heme Planarity and Accessibility in Horseradish Peroxidase

Some noncovalently linked hemes like those in the peroxidases have highly conserved characteristic distortions in the porphyrin plane. Conservation occurs even for some proteins with a large natural variation in the amino acid sequence. Thus it is reasonable to anticipate that nonplanar porphyrins and protein-induced changes in the planarity may provide a mechanism for protein modulation of biological properties.We previously reported that covalent modification of three accessible charged lysines (Lys-174, Lys-232, Lys-241) to the hydrophobic anthraquinolysine residues successfully improves electron transfer properties, catalytic efficiency, and stability of HRP.Here we report that this modification also alters the Heme planarity and accessibility. Using the known x-ray crystallographic structure of HRP, two 3D models of HRP representing the native and modified enzyme, were constructed. Molecular dynamics simulations showed that upon modification, the accessibility of the heme prosthetic group to the substrates increased from 39.6 to 99.7 A2. Moreover, the Heme Planarity also affected significantly. While in the native structure the heme plane is concave and curves toward the proximal His, in the modified enzyme the heme assumes a planar conformation and shifts the position of the proximal His. This change causes the heme iron to become more exposed on the distal side of the porphyrin plane and so more accessible to the peroxide substrate. This result agrees well with the experimentally observed enhanced reactivity of the modified enzyme to the peroxide substrates.

Structure and Ligand Selection of Hemoglobin II from Lucina pectinata

Lucina pectinata ctenidia harbor three heme proteins: sulfide-reactive hemoglobin I (HbI(Lp)) and the oxygen transporting hemoglobins II and III (HbII(Lp) and HbIII(Lp)) that remain unaffected by the presence of H(2)S. The mechanisms used by these three proteins for their function, including ligand control, remain unknown. The crystal structure of oxygen-bound HbII(Lp) shows a dimeric oxyHbII(Lp) where oxygen is tightly anchored to the heme through hydrogen bonds with Tyr(30)(B10) and Gln(65)(E7). The heme group is buried farther within HbII(Lp) than in HbI(Lp). The proximal His(97)(F8) is hydrogen bonded to a water molecule, which interacts electrostatically with a propionate group, resulting in a Fe-His vibration at 211 cm(-1). The combined effects of the HbII(Lp) small heme pocket, the hydrogen bonding network, the His(97) trans-effect, and the orientation of the oxygen molecule confer stability to the oxy-HbII(Lp) complex. Oxidation of HbI(Lp) Phe(B10) --> Tyr and HbII(Lp) only occurs when the pH is decreased from pH 7.5 to 5.0. Structural and resonance Raman spectroscopy studies suggest that HbII(Lp) oxygen binding and transport to the host bacteria may be regulated by the dynamic displacements of the Gln(65)(E7) and Tyr(30)(B10) pair toward the heme to protect it from changes in the heme oxidation state from Fe(II) to Fe(III).

Resonance Raman Spectroscopy of Nitric Oxide Reductase and cbb3 Heme-Copper Oxidase

Elucidating the structure and properties of the active sites in cbb3 heme-copper oxidase and in nitric oxide reductase (Nor) is crucial in understanding the reaction mechanisms of oxygen and nitric oxide reduction by both enzymes. In the work here, we have applied resonance Raman (RR) spectroscopy to investigate the structure and properties of the binuclear heme b3-CuB center of cbb3 heme-copper oxidase from Pseudomonas stutzeri and the dinuclear heme b3-FeB center of Nor from Paracoccus denitrificans in the ligand-free and CO-bound forms and in the reactions with O2 and NO. The RR data demonstrate that in the Nor/NO reaction, the formation of the N-N bond occurs with the His-Fe heme b3 bond intact, and reformation of the heme b3-O-FeB dinuclear center causes the rupture of the proximal His-Fe heme b3 bond. In the reactions of Nor and cbb3 with O2, distinct oxidized heme b3 species, which differ from the as-isolated oxidized forms, have been characterized. The activation and reduction of O2 and NO by cbb3 oxidase and nitric oxide reductase are compared and discussed.

Narrow QRS Complex Tachycardia with Unusual Atrial Activation. What is the Mechanism?

Figure 1. Panel A shows the end of an entrainment run from the RV. From top to bottom: leads I, II, and V1 and recordings from the RA roof, anterior RA from high (A1) to low (A4), septal RA from low (S4) to high (S1), CS from proximal (CS1) to distal (CS5) and right pulmonary artery (RPA). Panel B shows the effect of an RV extrastimulus with proximal (His P) and distal (His D) His-bundle area recordings. The right lower panel shows the approximate location of the atrial recording catheters. Case Report A 48-year-old man underwent aortic and mitral valve replacement in 1986 for rheumatic valve disease. In 2004 reoperation was needed for prosthetic thrombosis and a transeptal incision was made to access the left atrium. Before reoperation he had episodes of paroxysmal atrial fibrillation but after the new procedure palpitations were

The formation of hydrogen bond in the proximal heme pocket of HemAT-Bs upon ligand binding

HemAT-Bs is the heme-based O 2 sensor responsible for aerotaxis control in Bacillus subtilis. In this study, we measured the time-resolved resonance Raman spectra of full-length HemAT-Bs wild-type (WT) and Y133F in the deoxy form and the photoproduct after photolysis of CO-bound form. In WT, the ν Fe–His band for the 10 ps photoproduct was observed at higher frequency by about 2 cm −1 compared with that of the deoxy form. This frequency difference is relaxed in hundreds of picoseconds. This time-dependent frequency shift would reflect the conformational change of the protein matrix. On the other hand, Y133F mutant did not show such a substantial ν Fe–His frequency shift after photolysis. Since a hydrogen bond to the proximal His induces an up-shift of the ν Fe–His frequency, these results indicate that Tyr133 forms a hydrogen bond to the proximal His residue upon the ligand binding. We discuss a functional role of this hydrogen bond formation for the signal transduction in HemAT-Bs.

Systematic Regulation of the Enzymatic Activity of Phenylacetaldoxime Dehydratase by Exogenous Ligands

Phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB) contains a heme that acts as the active site for the dehydration reaction of aldoxime. Ferrous heme is the active form, in which the heme is five coordinate with His282 as a proximal ligand. In this work, we evaluated the functional role of the proximal ligand for the catalytic properties of the enzyme by "the cavity mutant technique". The H282G mutant of OxdB lost enzymatic activity, although the heme, which was five coordinate with a water molecule (or OH-) as an axial ligand, existed in the protein matrix. The enzymatic activity was rescued by imidazole or pyridine derivatives that acted as the exogenous proximal ligand. By changing the electron-donation ability of the exogenous ligand with different substituents, the enzymatic activity could be regulated systematically. The stronger the electron-donation ability of the exogenous ligand, the higher was the restored enzymatic activity. Interestingly, H282G OxdB with 2-methyl imidazole showed a higher activity than the wild-type enzyme. Kinetic analyses revealed that the proximal His regulated not only the affinity of substrate binding to the heme but also the elimination of the OH group from the substrate.

Pathway of Information Transmission from Heme to Protein upon Ligand Binding/Dissociation in Myoglobin Revealed by UV Resonance Raman Spectroscopy

Gas sensory heme proteins respond to their environment by binding a specific gas molecule to heme and transmitting this primary binding signal to the protein. How the binding signal is transmitted from the heme to the protein remains to be clarified. Using UV resonance Raman (UVRR) spectroscopy, we investigated this pathway in sperm whale myoglobin as a model gas sensory heme protein. Based on the UVRR data and the effects of deleting one of three important pathways (His-93, 6-propionate, or 7-propionate), we determined the changes in the conformation of globin that occur upon binding of CO, nitric oxide (NO), or O(2) to heme and how they are transmitted from heme to globin. The UVRR results show that heme discriminates different ligands, resulting in different conformations in the globin protein. Specifically, NO induces changes in the spectrum of Trp residues in the A-helix that are significantly different from those induced by O(2) or CO binding. On the other hand, binding of O(2) to heme produces changes in the Tyr residues of the H-helix that are different from those induced by CO or NO binding. Furthermore, we found that cleavage of the Fe-His-93 covalent bond eliminates communication to the terminal region of the H-helix and that the 7-propionate hydrogen-bonding network is essential for transmitting the CO or NO binding signal to the N and C termini. Finally, the 6-propionate is important only for NO binding. Thus, the hydrogen-bonding network in the protein appears to be critical for intramolecular signal transduction in gas sensory heme proteins.

Spectroscopic characterization of the ferric states of Amphitrite ornata dehaloperoxidase and Notomastus lobatus chloroperoxidase: His-ligated peroxidases with globin-like proximal and distal properties

Amphitrite ornata dehaloperoxidase (DHP) and Notomastus lobatus chloroperoxidase (NCPO) catalyze the peroxide-dependent dehalogenation of halophenols and halogenation of phenols, respectively. Both enzymes have histidine (His) as their proximal heme iron ligand. Crystallographic examination of DHP revealed that it has a globin fold [M.W. LaCount, E. Zhang, Y.-P. Chen, K. Han, M.M. Whitton, D.E. Lincoln, S.A. Woodin, L. Lebioda, J. Biol. Chem. 275 (2000) 18712–18716] and kinetics studies established that ferric DHP is the active state [R.L. Osborne, L.O. Taylor, K. Han, B. Ely, J.H. Dawson, Biochem. Biophys. Res. Commun. 324 (2004) 1194–1198]. NCPO likely has these same properties. Previous work with His-ligated heme proteins has revealed characteristic spectral distinctions between dioxygen binding globins and peroxide-activating peroxidases. Since DHP, and likely NCPO, is a peroxide-activating globin, we have sought to determine in the present investigation whether the ferric resting states of these two novel heme-containing enzymes are myoglobin-like or peroxidase-like. To do so, we have examined their exogenous ligand-free ferric states as well as their azide, imidazole and NO bound ferric adducts (and ferrous–NO complexes) with UV–Visible absorption and magnetic circular dichroism spectroscopy. We have also compared each derivative to the analogous states of horse heart myoglobin (Mb) and horseradish peroxidase (HRP). The spectra observed for parallel forms of DHP and NCPO are virtually identical to each other as well as to the spectra of the same Mb states, while being less similar to the spectra of corresponding HRP derivatives. From these data, we conclude that exogenous ligand-free ferric DHP and NCPO are six-coordinate with water and neutral His as ligands. This coordination structure is distinctly different from the ferric resting state of His-ligated peroxidases and indicates that DHP and NCPO do not activate bound peroxide through a mechanism dependent on a push effect imparted by a partially ionized proximal His as proposed for typical heme peroxidases.

Residue F4 Plays a Key Role in Modulating Oxygen Affinity and Cooperativity in Scapharca Dimeric Hemoglobin

Residue F4 (Phe 97) undergoes the most dramatic ligand-linked transition in Scapharca dimeric hemoglobin, with its packing in the heme pocket in the unliganded (T) state suggested to be a primary determinant of its low affinity. Mutation of Phe 97 to Leu (previously reported), Val, and Tyr increases oxygen affinity from 8- to 100-fold over that of the wild type. The crystal structures of F97L and F97V show side chain packing in the heme pocket for both R and T state structures. In contrast, in the highest-affinity mutation, F97Y, the tyrosine side chain remains in the interface (high-affinity conformation) even in the unliganded state. Comparison of these mutations reveals a correlation between side chain packing in the heme pocket and oxygen affinity, indicating that greater mass in the heme pocket lowers oxygen affinity due to impaired movement of the heme iron into the heme plane. The results indicate that a key hydrogen bond, previously hypothesized to have a central role in regulation of oxygen affinity, plays at most only a small role in dictating ligand affinity. Equivalent mutations in sperm whale myoglobin alter ligand affinity by only 5-fold. The dramatically different responses to mutations at the F4 position result from subtle, but functionally critical, stereochemical differences. In myoglobin, an eclipsed orientation of the proximal His relative to the A and C pyrrole nitrogen atoms provides a significant barrier for high-affinity ligand binding. In contrast, the staggered orientation of the proximal histidine found in liganded HbI renders its ligand affinity much more susceptible to packing contacts between F4 and the heme group. These results highlight very different strategies used by cooperative hemoglobins in molluscs and mammals to control ligand affinity by modulation of the stereochemistry on the proximal side of the heme.

Heme Oxidation in a Chimeric Protein of the α-Selective Neisseriae meningitidis Heme Oxygenase with the Distal Helix of the δ-Selective Pseudomonas aeruginosa

Heme oxygenases from the bacterial pathogens Neisseriae meningitidis (nm-HO) and Pseudomonas aeruginosa (pa-HO) share significant sequence identity (37%). In nm-HO, biliverdin IXalpha is the sole product of the reaction, whereas pa-HO yields predominantly biliverdin IXdelta. We have previously shown by NMR that the in-plane conformation of the heme in pa-HO is significantly different from that of nm-HO as a result of distinct interactions of the heme propionates with the protein scaffold [Caignan, G. A., Deshmukh, R., Wilks, A., Zeng, Y., Huang, H. W., Moenne-Loccoz, P., Bunce, R. A., Eastman, M. A., and Rivera, M. (2002) J. Am. Chem. Soc. 124, 14879-14892]. In the report presented here, we have extended these studies to investigate the role of the distal helix by preparing a chimera of nm-HO (nm-HOch), in which distal helix residues 107-142 of nm-HO have been replaced with the corresponding residues of the delta-regioselective pa-HO (112-147). Electronic absorption spectra, resonance Raman and FTIR spectroscopic studies confirm that the orientation and hydrogen bonding properties of the proximal His ligand are not significantly altered in the chimera relative those of the wild-type proteins. The catalytic turnover of the nm-HOch-heme complex yields almost exclusively alpha-biliverdin and a small but reproducible amount of delta-biliverdin. NMR spectroscopic studies reveal that the altered regioselectivity in the chimeric protein likely stems from a dynamic equilibrium between two alternate in-plane conformations of the heme (in-plane heme disorder). Replacement of K16 with Ala and Met31 with Lys in the chimeric protein in an effort to tune key polypeptide-heme propionate contacts largely stabilizes the in-plane conformer conducive to delta-meso hydroxylation.

Peroxidase Activity of the Hemeoctapeptide N-Acetylmicroperoxidase-8

The pH dependence of the peroxidase activity (guaiacol assay) of the ferric hemeoctapeptide N-acetylmicroperoxidase-8 (N-AcMP8) was studied under conditions where formation of the Compound I analogue of the peroxidase enzymes is rate limiting. The pH profile of the reaction rate is consistent with a mechanism where both H2O2 and HO2- can displace H2O coordinated trans to neutral His but where the hydroxo complex of the hemepeptide (OH- trans to His) is kinetically inert. At pH > 11, where the proximal His ligand of Fe(III) ionizes to form a histidinate, the hydroxo complex (OH- trans to His-) becomes kinetically labile. A comparison of DeltaH(double dagger) and DeltaS(double dagger) values for the reaction of H2O2 and HO2-, obtained from the temperature dependence of the rate constants, with values for CN- and cysteine reported previously, shows that the activation parameters depend on the identity of the incoming ligand. This suggests that ligand substitution at Fe(III) in N-AcMP8 proceeds through an interchange mechanism.

Ventriculatrial Block During Atrioventricular Nodal Reentrant Tachycardia Suggesting Existence of an Upper Common Pathway

Studies on the mechanisms of atrioventricular nodal reentrant tachycardia (AVNRT) have yet to clarify whether the slow and fast pathways connect directly with the atria or via an upper common pathway. Although a "final common pathway" connecting the slow and fast pathways to the proximal His bundle was thought to be part of the reentrant circuit, debate on the presence of an upper common pathway continues. We report a case of AVNRT continuing despite the occurrence of ventriculoatrial block, thus supporting the existence of an upper common pathway.

Modeling the tertiary structure of a maize ( Zea mays ssp. mays) non-symbiotic hemoglobin

The tertiary structure of a maize ( Zea mays ssp. mays) non-symbiotic hemoglobin (Hbm) was modeled using computer tools and the known tertiary structure of rice Hb1 as a template. This method was tested by predicting the tertiary structure of soybean leghemoglobin a (Lba) using rice Hb1 as a template. The tertiary structures of the predicted and native Lba were similar, indicating that our computer methods could reliably predict the tertiary structures of plant Hbs. We next predicted the tertiary structure of Hbm. Hbm appears to have a long pre-helix A and a large CD-loop. The positions of the distal and proximal His are identical in Hbm and rice Hb1, which suggests that heme-Fe is hexacoordinate in Hbm and that the kinetic properties of Hbm and rice Hb1 are expected to be very similar, i.e. that Hbm has a high O 2-affinity. Thermostability analysis showed that Hbm CD-loop is unstable and may provide mobility to amino acids located at the heme pocket for both ligand binding and stabilization and heme-Fe coordination. Analysis of the C-terminal half of Hbm showed the existence of a pocket-like region (the N/C cavity) where interactions with organic molecules or proteins could be possible. Lys K94 protrudes into the N/C cavity, suggesting that K94 may sense the binding of molecules to the N/C cavity. Thus, it is likely that the instability of the CD-loop and the possibility of binding molecules to the N/C cavity are essential for positioning amino acids in the heme pocket and in regulating Hbm activity and function.

Photodissociation of Heme Distal Methionine in Ferrous Cytochrome c Revealed by Subpicosecond Time-Resolved Resonance Raman Spectroscopy

Cytochrome c (cyt c) is an electron-transfer heme protein that also binds nitric oxide (NO). In resting cyt c, two endogenous ligands of the heme iron are histidine-18 (His) and methionine-80 (Met) side chains, and NO binding requires the cleavage of one of the axial bonds. Previous femtosecond transient absorption studies suggested the photolysis of either Fe-His or Fe-Met bonds. We aimed at unequivocally identifying the internal side chain that is photodissociated in ferrous cyt c and at monitoring heme structural dynamics, by means of time-resolved resonance Raman (TR3) spectroscopy with approximately 0.6 ps time resolution. The Fe-His stretching mode at 216 cm-1 has been observed in photoproduct TR3 spectra for the first time for a c-type heme. The same transient mode was observed for a model ferrous cyt c N-fragment (residues 1-56) ligated with two His in the resting state. Our TR3 data reveal that upon ferrous cyt c photoexcitation, (i) distal Met side chain is instantly released, producing a five-coordinated domed heme structure, (ii) proximal His side chain, coupled to the heme, exhibits distortion due to strain exerted by the protein, and (iii) alteration in heme-cysteine coupling takes place along with the relaxation of the protein-induced deformations of the heme macrocycle.

The coordination of imidazole and substituted pyridines by the hemeoctapeptide N-acetyl-ferromicroperoxidase-8 (Fe IINAcMP8)

The N-terminus acetylated ferric hemeoctapeptide from cytochrome c, N-acetylmicroperoxidase-8 (Fe III-NAcMP8) can be reduced by dithionite in aqueous solution to produce Fe II-NAcMP8. The UV–Vis spectrum has a broad Soret band and relatively poorly defined Q bands which is consistent with a mixture of a five-coordinate high spin species with His as the axial ligand and a six-coordinate, predominantly high spin species with His/H 2O as axial ligands. There are two spectroscopically observable p K as at 8.7 ± 0.1 and 10.9 ± 0.2 which are attributed to ionization of a heme propionic acid group and coordinated H 2O, respectively; a p K a ⩾14 is due to ionization of the proximal His ligand. Equilibrium constants were determined by UV–Vis spectrophotometry at 25.0 ± 0.2 °C and 0.5 M ionic strength (NaClO 4) for the coordination of imidazole and a number of substituted pyridines, and complement available data for the ferric hemepeptide, allowing a comparison to be made of the affinity of an iron porphyrin with Fe in the +2 and +3 oxidation states towards these ligands. Imidazole is coordinated more strongly by the ferric porphyrin (log K = 4.08) than by the ferrous porphyrin (log K = 3.40). The equilibrium constants for coordination of pyridines by the ferric and ferrous porphyrins increase and decrease, respectively, with increasing ligand basicity. Values determined by cyclic voltammetry show the same dependence on the identity of the ligand. In the ferric porphyrin, the stability of the complex increases with the basicity of the ligand and hence its ability to donate electron density onto the metal. In the case of the more electron rich ferrous porphyrin, greater stability occurs with pyridine ligands that have an electron withdrawing group and hence can accept electron density from the metal. This is consistent with the midpoint reduction potentials E 1/2 of the pyridine complexes determined by cyclic voltammetry; E 1/2 is linearly dependent on, and becomes more negative with an increase in, ligand basicity. Log K for coordination of pyridines by the ferrous hemepeptide correlates well with the energy of the ligand frontier orbital with π symmetry, suggesting that π-bonding effects are significant in determining the strength of binding of pyridines by a ferrous porphyrin.