Heme Binding in Gas-Phase Holo-Myoglobin Cations: Distal Becomes Proximal?

His64 and His93 are the two well-known sites of heme binding in water-dissolved holo-myoglobin, with His93 being a proximal, strongly binding partner, while the distal His64 weakly coordinates to the heme through a small-molecule ligand, e.g., water or O₂. The heme bonding scheme in a water-free environment is as yet unclear. Here we employed electron transfer dissociation tandem mass spectrometry to study the preferential attachment site of the ferri-heme (Fe³⁺) in electrospray-produced 12+, 14+, and 16+ holo-myoglobin ions. Contrary to expectations, in lower-charge complexes that should have a structure resembling that in solution, the heme seems to be preferentially attached to the “distal” histidine. In contrast, in the highest studied charge state, the “proximal” histidine is the site of preferential attachment; the 14+ charge state is an intermediate case. This surprising finding raises a question of heme coordination in proteins transferred to water-free environment, as well as the effect of the protonation sites on heme bonding.     

The H93G Myoglobin Cavity Mutant as a Versatile Scaffold for Modeling Heme Iron Coordination Structures in Protein Active Sites and Their Characterization with Magnetic Circular Dichroism Spectroscopy

Preparation of heme model complexes is a challenging subject of long-standing interest for inorganic chemists. His93Gly sperm whale myoglobin (H93G Mb) has the proximal His replaced with the much smaller non-coordinating Gly. This leaves a cavity on the proximal side of the heme into which a wide variety of exogenous ligands can be delivered. The end result is a remarkably versatile scaffold for the preparation of model heme adducts to mimic the heme iron coordination structure of native heme proteins. In this review, we first summarize the quantitative evidence for differential ligand binding affinities of the proximal and distal pockets of the H93G Mb cavity mutant that facilitates the preparation of mixed-ligand derivatives. Then we review our use of magnetic circular dichroism and electronic absorption spectroscopy to characterize nitrogen-, oxygen-, and sulfur-donor-ligated H93G Mb adducts with an emphasis on species not easily prepared by other heme model system approaches and those that serve as spectroscopic models for native heme proteins.     

The hydrogen-bonding network in heme oxygenase also functions as a modulator of enzyme dynamics: chaotic motions upon disrupting the H-bond network in heme oxygenase from Pseudomonas aeruginosa

Relaxation compensated Carr-Purcell-Meiboom-Gill (rc-CPMG) NMR experiments have been used to investigate micros-ms motions in heme oxygenase from Pseudomonas aeruginosa (pa-HO) in its ferric state, inhibited by CN- (pa-HO-CN) and N3- (pa-HO-N3), and in its ferrous state, inhibited by CO (pa-HO-CO). Comparative analysis of the data from the three forms indicates that the nature of the coordinated distal ligand affects the micros-ms conformational freedom of the polypeptide in regions of the enzyme far removed from the heme iron and distal ligand. Interpretation of the dynamical information in the context of the crystal structure of resting state pa-HO shows that residues involved in the network of structural hydrogen-bonded waters characteristic of HOs undergo micros-ms motions in pa-HO-CN, which was studied as a model of the highly paramagnetic S = 5/2 resting state form. In comparison, similar motions are suppressed in the pa-HO-CO and pa-HO-N3 complexes, which were studied as mimics of the obligatory oxyferrous and ferric hydroperoxide intermediates, respectively, in the catalytic cycle of heme degradation. These findings suggest that in addition to proton delivery to the nascent Fe(III)-OO(-) intermediate during catalysis, the hydrogen-bonding network serves two additional roles: (i) propagate the electronic state (reactive state) in each of the distinct steps of the catalytic cycle to key but remote sections of the polypeptide via small rearrangements in the network of hydrogen bonds and (ii) modulate the conformational freedom of the enzyme, thus allowing it to adapt to the demanding changes in axial coordination state and substrate transformations that take place during the catalytic cycle. This idea was probed by disrupting the hydrogen-bonding network in pa-HO by replacing R80 with L. NMR spectroscopic studies conducted with R80L-pa-HO-N3 and R80L-pa-HO-CO revealed that the mutant exhibits nearly global conformational disorder, which is absent in the equivalent complexes of the wild type enzyme. The "chaotic" disorder in the R80L mutant is likely related to its significantly lower efficiency to hydroxylate heme in the presence of H2O2, relative to the wild type enzyme.     

The origin of stark splitting in the initial photoproduct state of MbCO

Ligand migration and binding in heme proteins have been measured by X-ray diffraction and time-resolved spectroscopy of photoproduct intermediates. In myoglobin (Mb), internal cavities serve as docking sites for carbon monoxide (CO) ligands. In these sites, the CO ligands display characteristic infrared (IR) stretching bands due to interactions with the local electrical field. In the primary docking site, a CO can reside in two opposite orientations, characterized by a doublet of infrared bands, B1 at approximately 2130 and B2 at approximately 2120 cm-1. To assign these bands to the specific orientations, we have reexamined the effects of mutating His64 and Val68 on the infrared stretching bands associated with the B1 and B2 photoproduct states. Wild-type, H64L, V68F, and H64L-V68F MbCO were selected for experimental and theoretical analyses. Fourier transform infrared (FTIR) spectroscopy and density functional theory (DFT) calculations were used to interpret the effects of the electrostatic environment on the B state bands. The imidazole side chain of His64 appears to be the primary cause of the observed Stark splitting. The high-frequency B1 band is assigned to the CO orientation in which the carbon (white atom) is directed toward the heme iron and the Nepsilon-H proton of His64. At low temperatures, CO molecules in the opposite orientational conformer, B2 with the O atom (red) toward His64, first rotate by 180 degrees into the more stable B1 state and then rebind.     

Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase

The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.     

Modulation of the ligand-field anisotropy in a series of ferric low-spin cytochrome c mutants derived from Pseudomonas aeruginosa cytochrome c-551 and Nitrosomonas europaea cytochrome c-552: a nuclear magnetic resonance and electron paramagnetic resonance study

Cytochromes of the c type with histidine-methionine (His-Met) heme axial ligation play important roles in electron-transfer reactions and in enzymes. In this work, two series of cytochrome c mutants derived from Pseudomonas aeruginosa (Pa c-551) and from the ammonia-oxidizing bacterium Nitrosomonas europaea (Ne c-552) were engineered and overexpressed. In these proteins, point mutations were induced in a key residue (Asn64) near the Met axial ligand; these mutations have a considerable impact both on heme ligand-field strength and on the Met orientation and dynamics (fluxionality), as judged by low-temperature electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectra. Ne c-552 has a ferric low-spin (S = 1/2) EPR signal characterized by large g anisotropy with g(max) resonance at 3.34; a similar large g(max) value EPR signal is found in the mitochondrial complex III cytochrome c1. In Ne c-552, deletion of Asn64 (NeN64Delta) changes the heme ligand field from more axial to rhombic (small g anisotropy and g(max) at 3.13) and furthermore hinders the Met fluxionality present in the wild-type protein. In Pa c-551 (g(max) at 3.20), replacement of Asn64 with valine (PaN64V) induces a decrease in the axial strain (g(max) at 3.05) and changes the Met configuration. Another set of mutants prepared by insertion (ins) and/or deletion (Delta) of a valine residue adjacent to Asn64, resulting in modifications in the length of the axial Met-donating loop (NeV65Delta, NeG50N/V65Delta, PaN50G/V65ins), did not result in appreciable alterations of the originally weak (Ne c-552) or very weak (Pa c-551) axial field but had an impact on Met orientation, fluxionality, and relaxation dynamics. Comparison of the electronic fingerprints in the overexpressed proteins and their mutants reveals a linear relationship between axial strain and average paramagnetic heme methyl shifts, irrespective of Met orientation or dynamics. Thus, for these His-Met axially coordinated Fe(III), the large g(max) value EPR signal does not represent a special case as is observed for bis-His axially coordinated Fe(III) with the two His planes perpendicular to each other.     

Ligand-field dependence of the excited state dynamics of Hangman bisporphyrin dyad complexes

A new Hangman porphyrin architecture has been developed to interrogate the ligand-field dependence of photoinduced PCET versus excitation energy transfer and intersystem crossing in PZn(II)-PFe(III)-OH dyads (P = porphyrin). In this design, a hanging carboxylic acid group establishes a hydrogen-bonding network to anchor the weak-field OH- ligand in the distal site of the PFe(III)-OH acceptor, whereas the proximal site is left available to accept strong-field imidazole ligands. Thus, controlling the tertiary coordination environment gives access to the first synthetic example of a porphyrin dyad with a biologically relevant weak-field/strong-field configuration of axial ligands at the heme. Transient absorption spectroscopy has been employed to probe the fate of the initial PZn(II)-based S1 excited state, revealing rapid S1 quenching for all dyads in the presence and absence of strong-field imidazole ligands (tau = 6-50 ps). The absence of a (P*+)Zn(II) signal that would complement photoinduced PCET at the PFe(III)-OH subunit (i.e., PFe(III)-OH --> PFe(II)-OH2) shows that excitation energy transfer and intersystem crossing channels dominate the quenching, regardless of whether proximal strong field ligands are present. Moreover, this photophysical assignment is independent of the solvent dielectric constant and whether a phenylene or biphenylene spacer is used to span the two porphyrin subunits. Electronic structure calculations suggest that the structural reorganization attendant to reductive PCET at the high-spin Fe(III)-OH center imposes a severe kinetic cost that can only be alleviated by inducing a low-spin electronic configuration with two strong-field axial ligands.     

Alkylamine-ligated H93G myoglobin cavity mutant: a model system for endogenous lysine and terminal amine ligation in heme proteins such as nitrite reductase and cytochrome f

His93Gly sperm whale myoglobin (H93G Mb) has the proximal histidine ligand removed to create a cavity for exogenous ligand binding, providing a remarkably versatile template for the preparation of model heme complexes. The investigation of model heme adducts is an important way to probe the relationship between coordination structure and catalytic function in heme enzymes. In this study, we have successfully generated and spectroscopically characterized the H93G Mb cavity mutant ligated with less common alkylamine ligands (models for Lys or the amine group of N-terminal amino acids) in numerous heme iron states. All complexes have been characterized by electronic absorption and magnetic circular dichroism spectroscopy in comparison with data for parallel imidazole-ligated H93G heme iron moieties. This is the first systematic spectral study of models for alkylamine- or terminal amine-ligated heme centers in proteins. High-spin mono- and low-spin bis-amine-ligated ferrous and ferric H93G Mb adducts have been prepared together with mixed-ligand ferric heme complexes with alkylamine trans to nitrite or imidazole as heme coordination models for cytochrome c nitrite reductase or cytochrome f, respectively. Six-coordinate ferrous H93G Mb derivatives with CO, NO, and O(2) trans to the alkylamine have also been successfully formed, the latter for the first time. Finally, a novel high-valent ferryl species has been generated. The data in this study represent the first thorough investigation of the spectroscopic properties of alkylamine-ligated heme iron systems as models for naturally occurring heme proteins ligated by Lys or terminal amines.     

A distal pocket Leu residue inhibits the binding of O2 and NO at the distal heme site of cytochrome c'

Cytochromes c' are pentacoordinate heme proteins with sterically hindered distal sites that bind NO and CO but do not form stable complexes with O(2). Removal of distal pocket steric hindrance via a Leu→Ala mutation yields favorable O(2) binding (K(d) ~49 nM) without apparent H-bond stabilization of the Fe-O(2) moiety, as well as an extremely high distal heme-NO affinity (K(d) ~70 fM). The native Leu residue inhibits distal coordination of diatomic ligands by decreasing k(on) as well as increasing k(off). The connection between distal steric constraints, k(off) values, and distal to proximal heme-NO conversion is discussed.     

Spectroscopic characterization and assignment of reduction potentials in the tetraheme cytochrome C554 from Nitrosomonas europaea

The tetraheme cytochrome c(554) (cyt c(554)) from Nitrosomonas europaea is an essential electron transfer component in the biological oxidation of ammonia. The protein contains one 5-coordinate heme and three bis-His coordinated hemes in a 3D arrangement common to a newly characterized class of multiheme proteins. The ligand binding, electrochemical properties, and heme-heme interactions are investigated with M?ssbauer and X- and Q-band (parallel/perpendicular mode) EPR spectroscopy. The results indicate that the 5-coordinate heme will not bind the common heme ligands, CN(-), F(-), CO, and NO in a wide pH range. Thus, cyt c(554) functions only in electron transfer. Analysis of a series of electrochemically poised and chemically reduced samples allows assignment of reduction potentials for heme 1 through 4 of +47, +47, -147, and -276 mV, respectively. The spectroscopic results indicate that the hemes are weakly exchange-coupled (J approximately -0.5 cm(-)(1)) in two separate pairs and in accordance with the structure: hemes 2/4 (high-spin/low-spin), hemes 1/3 (low-spin/low-spin). There is no evidence of exchange coupling between the pairs. A comparison of the reduction potentials between homologous hemes of cyt c(554) and other members of this new class of multiheme proteins is discussed. Heme 1 has a unique axial N(delta)-His coordination which contributes to a higher potential relative to the homologous hemes of hydroxylamine oxidoreductase (HAO) and the split-Soret cytochrome. Heme 2 is 300 mV more positive than heme 4 of HAO, which is attributed to hydroxide coordination to heme 4 of HAO.     

Ibuprofen induces an allosteric conformational transition in the heme complex of human serum albumin with significant effects on heme ligation

Human serum albumin (HSA), the most prominent protein in blood plasma, is able to bind a wide range of endogenous and exogenous compounds. Among the endogenous ligands, HSA is a significant transporter of heme, the heme-HSA complex being present in blood plasma. Drug binding to heme-HSA affects allosterically the heme affinity for HSA and vice versa. Heme-HSA, heme, and their complexes with ibuprofen have been characterized by electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopy. Comparison of the results for the heme and heme-HSA systems has provided insight into the structural consequences on the heme pocket of ibuprofen binding. The pentacoordinate tyrosine-bound heme coordination of heme-HSA, observed in the absence of ibuprofen, becomes hexacoordinate low spin upon ibuprofen binding, and heme dissociates at increasing drug levels. The electronic absorption spectrum and nu(Fe-CO)/nu(CO) vibrational frequencies of the CO-heme-HSA-ibuprofen complex, together with the observation of a Fe-His Raman mode at 218 cm(-1) upon photolysis of the CO complex and the low spin EPR g values indicate that a His residue is one of the low spin axial ligands, the sixth ligand probably being Tyr161. The only His residue in the vicinity of the heme Fe atom is His146, 9 A distant in the absence of the drug. This indicates that drug binding to heme-HSA results in a significant rearrangement of the heme pocket, implying that the conformational adaptability of HSA involves more than the immediate vicinity of the drug binding site. As a whole, the present spectroscopic investigation supports the notion that HSA could be considered as the prototype of monomeric allosteric proteins.     

Direct-detected 13C NMR to investigate the iron(III) hemophore HasA

Hemophore HasA is a 19 kDa iron(III) hemoprotein that participates in the shuttling of heme to a specific membrane receptor. In HasA, heme iron has an original coordination environment with a His/Tyr pair as axial ligands. Recently developed two-dimensional protonless (13)C-detected experiments provide the sequence-specific assignment of all but three protein residues in the close proximity of the paramagnetic center, thus overcoming limitations due to the short relaxation times induced by the presence of the iron(III) center. Mono-dimensional (13)C and (15)N experiments tailored for the detection of paramagnetic signals allow the identification of resonances of the axial ligands. These experiments are used to characterize the conformational features and the electronic structure of the heme iron(III) environment. The good complementarity among (1)H-, (13)C-, and (15)N-detected experiments is highlighted. A thermal high-spin/low-spin equilibrium is observed and is related to a modulation of the strength of the coordination bond between the iron and the Tyr74 axial ligand. The key role of a neighboring residue, His82, for the stability of the axial coordination and its involvement in the heme delivery to the receptor is discussed.     

Insight into heme protein redox potential control and functional aspects of six-coordinate ligand-sensing heme proteins from studies of synthetic heme peptides

We describe detailed studies of peptide-sandwiched mesohemes PSMA and PSMW, which comprise two histidine (His)-containing peptides covalently attached to the propionate groups of iron mesoporphyrin II. Some of the energy produced by ligation of the His side chains to Fe in the PSMs is invested in inducing helical conformations in the peptides. Replacing an alanine residue in each peptide of PSMA with tryptophan (Trp) to give PSMW generates additional energy via Trp side chain-porphyrin interactions, which enhances the peptide helicity and stability of the His-ligated state. The structural change strengthened His-FeIII ligation to a greater extent than His-FeII ligation, leading to a 56-mV negative shift in the midpoint reduction potential at pH 8 (Em,8 value). This is intriguing because converting PSMA to PSMW decreased heme solvent exposure, which would normally be expected to stabilize FeII relative to FeIII. This and other results presented herein suggest that differences in stability may be at least as important as differences in porphyrin solvent exposure in governing redox potentials of heme protein variants having identical heme ligation motifs. Support for this possibility is provided by the results of studies from our laboratories comparing the microsomal and mitochondrial isoforms of mammalian cytochrome b5. Our studies of the PSMs also revealed that reduction of FeIII to FeII reversed the relative affinities of the first and second His ligands for Fe (K2III > K1III; K2II < K1II). We propose that this is a consequence of conformational mobility of the peptide components, coupled with the much greater ease with which FeII can be pulled from the mean plane of a porphyrin. An interesting consequence of this phenomenon, which we refer to as "dynamic strain", is that an exogenous ligand can compete with one of the His ligands in an FeII-PSM, a reaction accompanied by peptide helix unwinding. In this regard, the PSMs are better models of neuroglobin, CooA, and other six-coordinate ligand-sensing heme proteins than of stably bis(His)-ligated electron-transfer heme proteins such as cytochrome b5. Exclusive binding of exogenous ligands by the FeII form of PSMA led to positive shifts in its Em,8 value, which increases with increasing ligand strength. The possible relevance of this observation to the function of six-coordinate ligand-sensing heme proteins is discussed.     

The ferrous verdoheme-heme oxygenase complex is six-coordinate and low-spin

A biosynthetic and enzymatic method was developed for the preparation of 13C-labeled verdoheme, which permits the 13C NMR spectroscopic characterization of this elusive intermediate in the heme oxidation path catalyzed by the enzyme heme oxygenase. The 13C NMR data indicate that the ferrous verdoheme complex of Neisseria meningitides heme oxygenase is hexacoordinate and diamagnetic, with a proximal histidine and likely a distal hydroxide as axial ligands. The coordination number and spin state of the ferrous verdoheme-heme oxygenase complex is in stark contrast to the pentacoordinate and paramagnetic nature of the heme-heme oxygenase complex and heme centers in general.     

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.     

Unusual peroxidase activity of a myoglobin mutant with two distal histidines

By retaining the native distal His64 in sperm whale myoglobin(Mb),a second distal histidine was engineered in Mb by mutating Leu29 to His29.The resultant mutant of L29H Mb exhibits an unusual enhanced peroxidase activity with a positive cooperativity in comparison to that of wild type Mb.The new enzyme with two cooperative distal histidines has not been found in native peroxidase, which emphasizes a creation of the rational protein design.     

Weak-field anions displace the histidine ligand in a synthetic heme peptide but not in N-acetylmicroperoxidase-8: possible role of heme geometry differences

We have recently reported that aquo and thioether complexes of the ferric cytochrome c heme peptide N-acetylmicroperoxidase-8 (FeIII-1) exhibit greater low-spin character than do the corresponding complexes of a synthetic, water-soluble, monohistidine-ligated heme peptide (FeIII-2; Cowley, A. B.; Lukat-Rodgers, G. S.; Rodgers, K. R.; Benson, D. R. Biochemistry 2004, 43, 1656-1666). Herein we report results of studies showing that weak-field ligands bearing a full (fluoride, chloride, hydroxide) or partial (phenoxide, thiocyanate) negative charge on the coordinating atom trigger dissociation of the axial His ligand in FeIII-2 but not in FeIII-1. We attribute the greater sensitivity of His ligation in FeIII-1 to weak-field anionic ligands than to weak-field neutral ligands to the following phenomena: (1) anionic ligands pull FeIII further from the mean plane of a porphyrin than do neutral ligands, which will have the effect of straining the His-Fe bond in FeIII-2, and (2) heme in FeIII-2 is likely to undergo a modest doming distortion following anion binding that will render the His-ligated side of the porphyrin concave, thereby increasing porphyrin/ligand steric interactions. We propose that ruffling of the heme in FeIII-1 is an important factor contributing to its ability to resist His dissociation by weak-field anions. First, ruffling should allow His to more closely approach the porphyrin than is possible in FeIII-2, thereby reducing bond strain following anion binding. Second, the ruffling deformation in FeIII-1, which is enforced by the double covalent heme-peptide linkage, will almost certainly prevent significant porphyrin doming.     

Changes in the heme ligation during folding of a Geobacter sulfurreducens sensor GSU0935

Ligand binding and substitution reactions are important for metalloprotein folding and function. The heme sensor of a methyl-accepting chemotaxis GSU0935 is a c-type cytochrome from the bacterium Geobacter sulfurreducens. The heme domain switches one of its axial ligands from H(2)O to a low-spin ligand, presumably Met, upon reduction. The study analyzes the stability and folding kinetics of the ferric domain. Guanidine hydrochloride denaturation yields the low-spin heme species arising from coordination of the ferric heme by non-native His residues. The population of the low-spin species further increases and then declines during protein refolding. Kinetics and mutational effects suggest that His54, from the N-terminal region of the domain, is the transient ligand to the heme. The capture and release of a non-native ligand within the compact partially-folded structures illustrates the flexibility of the heme environment in GSU0935, which may relate to the domain sensor function.     

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.     

Role of heme types in heme-copper oxidases: effects of replacing a heme b with a heme o mimic in an engineered heme-copper center in myoglobin

To address the role of the secondary hydroxyl group of heme a/o in heme-copper oxidases, we incorporated Fe(III)-2,4 (4,2) hydroxyethyl vinyl deuterioporphyrin IX, as a heme o mimic, into the engineered heme-copper center in myoglobin (sperm whale myoglobin L29H/F43H, called Cu(B)Mb). The only difference between the heme b of myoglobin and the heme o mimic is the substitution of one of the vinyl side chains of the former with a hydroxyethyl group of the latter. This substitution resulted in an approximately 4 nm blue shift in the Soret band and approximately 20 mV decrease in the heme reduction potential. In a control experiment, the heme b in Cu(B)Mb was also replaced with a mesoheme, which resulted in an approximately 13 nm blue shift and approximately 30 mV decrease in the heme reduction potential. Kinetic studies of the heme o mimic-substituted Cu(B)Mb showed significantly different reactivity toward copper-dependent oxygen reduction from that of the b-type Cu(B)Mb. In reaction with O2, Cu(B)Mb with a native heme b showed heme oxygenase activity by generating verdoheme in the presence of Cu(I). This heme degradation reaction was slowed by approximately 19-fold in the heme o mimic-substituted Cu(B)Mb (from 0.028 s(-1) to 0.0015 s(-1)), while the mesoheme-substituted Cu(B)Mb shared a similar heme degradation rate with that of Cu(B)Mb (0.023 s(-1)). No correlation was found between the heme reduction potential and its O2 reactivity. These results strongly suggest the critical role of the hydroxyl group of heme o in modulating heme-copper oxidase activity through participation in an extra hydrogen-bonding network.