Heme pocket structural properties of a bacterial truncated hemoglobin from Thermobifida fusca

An acidic surface variant (ASV) of the "truncated" hemoglobin from Thermobifida fusca was designed with the aim of creating a versatile globin scaffold endowed with thermostability and a high level of recombinant expression in its soluble form while keeping the active site unmodified. This engineered protein was obtained by mutating the surface-exposed residues Phe107 and Arg91 to Glu. Molecular dynamics simulations showed that the mutated residues remain solvent-exposed, not affecting the overall protein structure. Thus, the ASV was used in a combinatorial mutagenesis of the distal heme pocket residues in which one, two, or three of the conserved polar residues [TyrB10(54), TyrCD1(67), and TrpG8(119)] were substituted with Phe. Mutants were characterized by infrared and resonance Raman spectroscopy and compared with the wild-type protein. Similar Fe-proximal His stretching frequencies suggest that none of the mutations alters the proximal side of the heme cavity. Two conformers were observed in the spectra of the CO complexes of both wild-type and ASV protein: form 1 with ν(FeC) and ν(CO) at 509 and 1938 cm(-1) and form 2 with ν(FeC) and ν(CO) at 518 and 1920 cm(-1), respectively. Molecular dynamics simulations were performed for the wild-type and ASV forms, as well as for the TyrB10 mutant. The spectroscopic and computational results demonstrate that CO interacts with TrpG8 in form 1 and interacts with both TrpG8 and TyrCD1 in form 2. TyrB10 does not directly interact with the bound CO.     

Resonance Raman evidence that distal histidine protonation removes the steric hindrance to upright binding of carbon monoxide by myoglobin

The resonance Raman band assigned to Fe--CO stretching in the sperm whale myoglobin CO adduct shifts from 507 cm-1 at neutral pH to 488 cm-1 at low pH, in concert with a shift of the C-O stretching infrared band from 1947 to 1967 cm-1 (Fuchsman & Appleby, 1979), while the 575-cm-1 Fe-C-O bending RR band loses intensity. The pKa that characterizes these changes is approximately 4.4. The vibrational frequencies at low pH are well modeled by the protein-free CO, imidazole adduct of protoheme in a nonpolar solvent while those at high pH are modeled by the adduct of a heme with a covalent strap (Yu et al., 1983) which inhibits upright CO binding. It is inferred that the Fe-C-O unit changes from a tilted to an upright geometry when the distal histidine is protonated, because its side chain swings out of the heme pocket due to electrostatic repulsion with a nearby arginine residue. A different protonation step (pKa = 5.7), which has been shown to modulate the CO rebinding kinetics (Doster et al., 1982) as well as the optical spectrum (Fuchsman & Appleby, 1979), is suggested to involve a global structure change associated with protonation of histidine residues distant from the heme.     

Two types of conformers with distinct Fe-C-O configuration in the ferrous CO complex of horseradish peroxidase. Resonance Raman and infarared spectroscopic studies with native and deuteroheme-substituted enzymes

The presence of at least two types of conformers in the ferrous CO complex of horseradish peroxidase has been demonstrated with the use of native and deuteroheme-substituted enzymes. Type I conformers, predominant in acidic pH, exhibited both an Fe-CO stretching and an Fe-C-O bending Raman line together with an infrared C-O stretch band below 1920 em-1. On the other hand, type II conformers, dominant species in alkaline pH, showed only an Fe-CO stretching Raman line with the C-O stretch above 1930 cm-1. They were interconvertible either by the changes in pH or by the binding of benzhydroxamate, a substrate for the enzyme. The pKa value for the pH-dependent interconversion of CO complex of deuteroheme-substituted enzyme was 8.3. These findings were interpreted to mean that the bound CO molecule in type I conformers was more tilted over the heme-plane than that in type II conformers. A steric hindrance by the bound substrate or the protonated form of a distal amino acid residue, presumably of histidine, is considered to be the cause for the isomerization. By summarizing present and previous data on the vibrational frequencies of heme-carbonyl complexes, we found that there are inverse-linear relationships between the square of Fe-CO and that of C-O stretching frequencies, while squares of Fe-CO stretching and Fe-C-O bending frequencies were linearly correlated with each other. Also found is that the dissociation rate constant of CO molecule from heme-carbonyl complexes is a linear function of the Fe-CO stretching frequency. The significance of these results is discussed.     

Effects of cholesterol side-chain groups and adrenodoxin binding on the vibrational modes of carbon monoxide bound to cytochrome P-450scc: implications of the productive and nonproductive substrate bindings.

Effects of the bindings of cholesterol and its hydroxylated analogues on the Fe-CO stretching and the C-O stretching vibrations of cytochrome P-450scc-CO complex were examined by resonance Raman and FT-IR spectroscopies to reveal the spatial relationship between the steroid side-chain groups and the heme-bound C-O moiety at the active center. These C-O and Fe-CO vibrations exhibited considerable variations depending on the steroids used; however, analyses on the nu Fe-CO vs nu C-O plot for cytochrome P-450scc indicated the absence of the negative correlation between these two vibrations, which is common among various Fe(2+)-porphyrin-CO complexes having imidazole ligands. Rather, we noticed the existence of two groups depending on substrates, the one exhibiting C-O infrared absorption bands in the region from 1930 to 1940 cm-1 and higher enzymatic turnover numbers in the reconstituted enzymatic systems and the other exhibiting C-O infrared absorption bands in the region above 1945 cm-1 and lower enzymatic turnover numbers. Thus, the former substrate group is likely to be fitted into the substrate binding site in the efficient "productive substrate binding" structure, whereas the latter group may be bound to the enzyme in the structure not suitable for the efficient enzymatic reaction ("nonproductive substrate binding" conformation).(ABSTRACT TRUNCATED AT 250 WORDS)     

The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.     

Resonance Raman detection of a v(Fe-CO) stretching frequency in cytochrome P-450scc from bovine adrenocortical mitochondria

Resonance Raman scattering experiments on CO-complexed cytochrome P-450scc from bovine adrenocortical mitochondria demonstrate the simultaneous enhancement of v(Fe-CO) stretching and bound v(C-O) stretching frequencies at 477 and 1953 cm-1, respectively. These assignments were made on the basis of frequency shifts with the isotope 12C18O. This unusually low v(Fe-CO) stretching frequency in cytochrome P-450scc, compared with other CO-complexed hemoproteins such as CO-hemoglobin and -myoglobin, is presumably due to the thiolate ligation to the heme iron trans to CO and due to the linear and perpendicular configuration of CO binding to the heme.     

Carbon monoxide adducts of KatG and KatG(S315T) as probes of the heme site and isoniazid binding

KatG, the catalase peroxidase from Mycobacterium tuberculosis, is important in the activation of the antitubercular drug, isoniazid. About 50% of isoniazid-resistant clinical isolates contain a mutation in KatG wherein the serine at position 315 is substituted with threonine, KatG(S315T). The heme pockets of KatG and KatG(S315T) and their interactions with isoniazid are probed using resonance Raman (rR) spectroscopy to characterize their ferrous CO complexes. Three vibrational modes, C-O and Fe-C stretching and Fe-CO bending, are assigned using 12CO and 13CO isotope shifts. Two conformers are observed for KatG-CO and KatG(S315T)-CO. Resonance Raman features assigned to form I are consistent with it having a neutral proximal histidine ligand and the Fe-C-O moiety hydrogen bonded to a distal residue. The nu(C-O) band for form I is sharp, consistent with a conformationally homogeneous Fe-CO unit. Form II also has a neutral proximal histidine ligand but is not hydrogen bonded. This appears to result in a conformationally disordered Fe-CO unit, as evidenced by a comparatively broad C-O stretching band. The 13CO-sensitive bands assigned to form II are predominant in the KatG(S315T)-CO rR spectrum. Isoniazid binding is apparent from the resonance Raman signatures of both WT KatG-CO and KatG(S315T)-CO. Moreover, isoniazid binding elicits an increase in the form I population of wild-type KatG-CO while having little, if any, effect on the already low population of form I of KatG(S315T)-CO. Since oxyKatG (compound III) also contains a low-spin diatomic ligand-heme adduct (heme-O2), it is reasonable to suggest that it too would exist as a mixture of conformers. Because the small form I population of KatG(S315T)-CO correlates with its inability to activate INH, we hypothesize that form I plays a role in INH activation.     

Effects of crystallization on the heme-carbon monoxide moiety of bovine heart cytochrome c oxidase carbonyl.

Cytochrome c oxidase isolated from bovine heart was crystallized in the fully reduced carbon monoxide (CO)-bound form. To evaluate the structure of the O2 reaction site in crystals and in solution, the bound C-O stretch infrared band in protein crystals was compared with the band for protein solution. In solution, the C-O stretch band could be deconvoluted into two extremely narrow bands, one at 1963.6 cm-1 with delta v1/2 = 3.4 cm-1 of 60% Gaussian/40% Lorentzian character represented 86% of the total band area and the other at 1960.3 cm-1 with delta v1/2 = 3.0 cm-1 of 47% Gaussian/53% Lorentzian character represented 14% of the total band area. The crystals exhibited two deconvoluted C-O infrared bands having very similar band parameters with those in solution. These findings support the presence of two structurally similar conformers in both crystals and solution. Thus crystallization of this enzyme does not affect the structure at the CO-binding site to as great extent as has been noted for myoglobin and hemoglobin carbonyls, indicating that the active (CO- or O2-binding) site of cytochrome c oxidase must be conformationally very stable and highly ordered compared to other hemoproteins such as hemoglobin.     

Resonance Raman investigation of CO-ligated monomeric insect hemoglobins. Direct evidence for reciprocal changes in iron-axial ligand bonds induced by allosteric transitions

The resonance Raman spectra of the two affinity states of the CO-ligated monomeric insect hemoglobins, Chironomus thummi thummi (CTT) III ad IV, have been investigated. We have identified (via 54Fe/57Fe and 13C18O/12C16O isotope exchange) the Fe-N epsilon(His) stretching mode at approximately 317 cm-1. This stretching mode changes from 329 (pH 5.5) to 317 cm-1 (pH 9.5) reflecting the pH-induced t in equilibrium with r conformational transition. The Fe-CO stretching mode is also pH-sensitive changing from 483 (pH 5.2) to 485 cm-1 (pH 9.2) in 57Fe CTT III . 13C18O complex. However the C-O stretching mode is pH-insensitive. The nonallosteric monomeric insect hemoglobin CTT I does not exhibit a pH-dependence of these vibrational modes. pH-Induced effects were also observed for a vinyl bending mode at 379 cm-1 (pH 9.5) in CTT III deuterated at the beta-carbons of the vinyls in position 2 and 4. It shifts to 390 cm-1 at pH 5.5. The other vinyl vibration at 573 cm-1 exhibits intensity enhancement via through-space coupling with the Fe-C-O bending mode. Our resonance Raman data provide the first direct evidence that the trans-effect is operative as a trigger mechanism for ligand-binding in monomeric allosteric insect hemoglobins. In going from the low-affinity to the high-affinity state, the Fe-N epsilon(His) bond becomes weaker, whereas the Fe-CO bond becomes stronger.     

Role of the distal hydrogen-bonding network in regulating oxygen affinity in the truncated hemoglobin III from Campylobacter jejuni

Oxygen affinity in heme-containing proteins is determined by a number of factors, such as the nature and conformation of the distal residues that stabilize the heme bound-oxygen via hydrogen-bonding interactions. The truncated hemoglobin III from Campylobacter jejuni (Ctb) contains three potential hydrogen-bond donors in the distal site: TyrB10, TrpG8, and HisE7. Previous studies suggested that Ctb exhibits an extremely slow oxygen dissociation rate due to an interlaced hydrogen-bonding network involving the three distal residues. Here we have studied the structural and kinetic properties of the G8(WF) mutant of Ctb and employed state-of-the-art computer simulation methods to investigate the properties of the O(2) adduct of the G8(WF) mutant, with respect to those of the wild-type protein and the previously studied E7(HL) and/or B10(YF) mutants. Our data indicate that the unique oxygen binding properties of Ctb are determined by the interplay of hydrogen-bonding interactions between the heme-bound ligand and the surrounding TyrB10, TrpG8, and HisE7 residues.     

Effect of the distal residues on the vibrational modes of the Fe-CO bond in hemoglobin studied by protein engineering

Using an Escherichia coli gene expression system, we have engineered human hemoglobin (Hb) mutants having the distal histidine (E7) and valine (E11) residues replaced by other amino acids. The interaction between the mutated distal residues and bound carbon monoxide has been studied by Soret-excited resonance Raman spectroscopy. The replacement of Val-E11 by Ala, Leu, Ile, and Met has no effect on the v(C-O), v(Fe-CO) stretching or delta(Fe-C-O) bending frequencies in both the alpha and beta subunits of Hb, although some of these mutations affect the CO affinity as much as 40-fold. The strain imposed on the protein by the binding of CO is not localized in the Fe-CO bond and is probably distributed among many bonds in the globin. The replacement of His-E7 by Val or Gly brings the stretching frequencies v(Fe-CO) and v(C-O) close to those of free heme complexes. In contrast, the substitution of His-E7 by Gln, which is flexible and polar, produces no effects on the resonance Raman spectrum of either alpha- or beta-globin. The replacement of His-E7 of beta-globin by Phe shows the same effect as replacement by Gly or Val. Therefore, the steric bulk of the distal residues is not the primary determinant of the Fe-CO ligand vibrational frequencies. The ability of both histidine and glutamine to alter the v(C-O), v(Fe-CO), or delta(Fe-C-O) frequencies may be attributed to the polar nature of their side chains which can interact with bound CO in a similar manner.     

Resonance Raman investigation of the effects of copper binding to iron-mesoporphyrin.histidine-rich glycoprotein complexes.

Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Raman bands indicative of low-spin, six-coordinate iron before Cu2+ binding exhibit monotonic intensity shifts to bands representing high-spin, five-coordinate iron. The latter coordination is in contrast to that found in hemoglobin and myoglobin, and explains the Cu(2+)-induced decrease and broadening of the Fe(3+)-mesoporphyrin.HRG Soret band concomitant with the increase in the high-spin marker band at 620 nm. After dithionite reduction, the Fe(2+)-mesoporphyrin.HRG complex displays high frequency resonance Raman bands characteristic of low-spin heme and no iron-histidine stretch, which together suggest six-coordinate iron. Furthermore, the local heme environment of the complex is not altered by the binding of Cu1+. CO-bound Fe(2+)-mesoporphyrin.HRG exhibits bands in the high and low frequency regions similar to those of other CO-bound heme proteins except that the iron-CO stretch at 505 cm-1 is unusually broad with delta nu approximately 30 cm-1. The dynamics of CO photolysis and rebinding to Fe(2+)-mesoporphyrin.HRG are also distinctive. The net quantum yield for photolysis at 10 ns is low relative to most heme proteins, which may be attributed to very rapid geminate recombination. A similar low net quantum yield and broad iron-CO stretch have so far only been observed in a dimeric cytochrome c' from Chromatium vinosum. Furthermore, the photolytic transient of Fe(2+)-mesoporphyrin.HRG lacks bands corresponding to high-spin, five-coordinate iron as is found in hemoglobin and myoglobin under similar experimental conditions, suggesting iron hexacoordination before CO recombination. These data are consistent with a closely packed distal heme pocket that hinders ligand diffusion into the surrounding solvent.     

Purification and spectroscopic characterization of Ctb, a group III truncated hemoglobin implicated in oxygen metabolism in the food-borne pathogen Campylobacter jejuni

Campylobacter jejuni is a food-borne bacterial pathogen that possesses two distinct hemoglobins, encoded by the ctb and cgb genes. The former codes for a truncated hemoglobin (Ctb) in group III, an assemblage of uncharacterized globins in diverse clinically and technologically significant bacteria. Here, we show that Ctb purifies as a monomeric, predominantly oxygenated species. Optical spectra of ferric, ferrous, O(2)- and CO-bound forms resemble those of other hemoglobins. However, resonance Raman analysis shows Ctb to have an atypical nu(Fe)(-)(CO) stretching mode at 514 cm(-)(1), compared to those of the other truncated hemoglobins that have been characterized so far. This implies unique roles in ligand stabilization for TyrB10, HisE7, and TrpG8, residues highly conserved within group III truncated hemoglobins. Because C. jejuni is a microaerophile, and a ctb mutant exhibits O(2)-dependent growth defects, one of the hypothesized roles of Ctb is in the detoxification, sequestration, or transfer of O(2). The midpoint potential (E(h)) of Ctb was found to be -33 mV, but no evidence was obtained in vitro to support the hypothesis that Ctb is reducible by NADH or NADPH. This truncated hemoglobin may function in the facilitation of O(2) transfer to one of the terminal oxidases of C. jejuni or, instead, facilitate O(2) transfer to Cgb for NO detoxification.     

Time-resolved FT-IR studies on the CO adduct of Paracoccus denitrificans cytochrome c oxidase: comparison of the fully reduced and the mixed valence form.

The rebinding of CO to cytochrome c oxidase from Paracoccus denitrificans in the fully reduced and in the half-reduced (mixed valence) form as a function of temperature was investigated using time-resolved rapid-scan FT-IR spectroscopy in the mid-IR (1200-2100 cm-1). For the fully reduced enzyme, rebinding was complete in approximately 2 s at 268 K and showed a biphasic reaction. At 84 K, nonreversible transfer of CO from heme a3 to CuB was observed. Both photolysis at 84 K and photolysis at 268 K result in FT-IR difference spectra which show similarities in the amide I, amide II, and heme modes. Both processes, however, differ in spectral features characteristic for amino acid side chain modes and may thus be indicative for the motional constraint of CO at low temperature. Rebinding of photodissociated CO for the mixed-valence enzyme at 268 K is also biphasic, but much slower as compared to the fully reduced enzyme. FT-IR difference spectra show band features similar to those for the fully reduced enzyme. Additional strong bands in the amide I and amide II range indicate local conformational changes induced by electron and coupled proton transfer. These signals disappear when the temperature is lowered to 84 K. At 268 K, a difference signal at 1746 cm-1 is observed which is shifted by 6 cm-1 to 1740 cm-1 in 2H2O. The absence of this signal for the mutant Glu 278 Gln allows assignment to the COOH stretching mode of Glu 278, and indicates changes of the conformation, proton position, or protonation of this residue upon electron transfer.     

Structural heterogeneity of the Fe(2+)-N epsilon (HisF8) bond in various hemoglobin and myoglobin derivatives probed by the Raman-active iron histidine stretching mode.

We have examined the Fe(2+)-N epsilon (HisF8) complex in hemoglobin A (HbA) by measuring the band profile of its Raman-active nu Fe-His stretching mode at pH 6.4, 7.0, and 8.0 using the 441-nm line of a HeCd laser. A line shape analysis revealed that the band can be decomposed into five different sublines at omega 1 = 195 cm-1, omega 2 = 203 cm-1, omega 3 = 212 cm-1, omega 4 = 218 cm-1, and omega 5 = 226 cm-1. To identify these to the contributions from the different subunits we have reanalyzed the nu Fe-His band of the HbA hybrids alpha(Fe)2 beta(Co)2 and alpha(Co)2 beta(Fe)2 reported earlier by Rousseau and Friedman (D. Rousseau and J. M. Friedman. 1988. In Biological Application on Raman Spectroscopy. T. G. Spiro, editor, 133-216). Moreover we have reanalyzed other Raman bands from the literature, namely the nu Fe-His band of the isolated hemoglobin subunits alpha SH- and beta SH-HbA, various hemoglobin mutants (i.e., Hb(TyrC7 alpha-->Phe), Hb(TyrC7 alpha-->His), Hb M-Boston and Hb M-Iwate), N-ethylmaleimide-des(Arg141 alpha) hemoglobin (NES-des(Arg141 alpha)HbA) and photolyzed carbonmonoxide hemoglobin (Hb*CO) measured 25 ps and 10 ns after photolysis. These molecules are known to exist in different quaternary states. All bands can be decomposed into a set of sublines exhibiting frequencies which are nearly identical to those found for deoxyhemoglobin A. Additional sublines were found to contribute to the nu Fe-His band of NES-des(Arg141 alpha) HbA and the Hb*CO species. The peak frequencies of the bands are determined by the most intensive sublines. Moreover we have measured the nu Fe-His band of deoxyHbA at 10 K in an aqueous solution and in a 80% glycerol/water mixture. Its subline composition at this temperature depends on the solvent and parallels that of more R-like hemoglobin derivatives. We have also measured the optical charge transfer band III of deoxyHbA at room temperature and found, that at least three subbands are required to fit its asymmetric band shape. This corroborates the findings on the nu Fe-His band in that it is indicative of a heterogeneity of the Fe(2+)-N epsilon(HisF8) bond. Finally we measured the nu Fe-His band of horse heart deoxyMb at different temperatures and decomposed it into three different sublines. In accordance with what was obtained for HbA their intensities rather than their frequencies are temperature-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structural characterization of the proximal and distal histidine environment of cytoglobin and neuroglobin

Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR3) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (nu(Fe-His)) bands at 229 and 221 cm(-1), respectively. No time-dependent shift in the nu(Fe-His) band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (nu(Fe-CO), nu(C-O), delta(Fe-C-O)) and (nu(Fe-NO), nu(N-O), delta(Fe-N-O)) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties.     

Resonance Raman study of cytochrome b562-o complex, a terminal oxidase of Escherichia coli in its ferric, ferrous, and CO-ligated states

Cytochrome b562-o complex, a terminal oxidase in the respiratory chain of aerobically grown Escherichia coli, has been studied by resonance Raman spectroscopy in its air-oxidized, dithionite-reduced, and reduced and CO-ligated states. In the reduced state, with a 406.7-nm excitation, there appeared 1494 and 1473 cm-1 lines, indicating that low spin and high spin components are included in the cytochrome b562-o complex. For the air-oxidized protein, resonance Raman lines were observed at 1372, 1503, and 1580 cm-1 with a 413.1-nm excitation, indicating that there is a ferric low spin heme. In addition, a weak but appreciable Raman line was observed at 1480 cm-1 assignable to a ferric high spin heme. Accordingly, it was concluded that low spin and high spin components are included in the cytochrome b562-o complex in the reduced and the air-oxidized states. In the CO-ligated state, with a defocused laser beam of 413.1 nm, two Raman bands assignable to the Fe-CO stretching mode have been observed at 489 and 523 cm-1, as a major and a minor component, respectively. When the laser beam was focused upon the sample to cause a photodissociation of CO from the heme moiety, the intensity of the major band at 489 cm-1 was reduced as expected. On the other hand, the minor band at 523 cm-1 remained still obvious. It was suggested that the cytochrome b562-o complex may have an additional anomalous site for CO that is resistant to photodissociation.     

Resonance raman investigations of site-directed mutants of myoglobin: effects of distal histidine replacement

The resonance Raman spectra of met-, deoxy-, and (carbonmonoxy)myoglobin (MbCO) are studied as a function of amino acid replacement at the distal histidine-E7 position. The synthetic wild type is found to be spectroscopically identical with the native material. The methionine and glycine replacements do not affect the met or deoxy spectra but do lead to distinct changes in the nu Fe-CO region of the MbCO spectrum. The native MbCO displays a pH-dependent population redistribution of the nu Fe-CO modes, while the analogous population in the mutant systems is found to be pH independent. This indicates that histidine-E7 is the titratable group in native MbCO. Moreover, the pH dependence of the population dynamics is found to be inconsistent with a simple two-state Henderson-Hasselbalch analysis. Instead, we suggest a four-state model involving the coupling of histidine protonation and conformational change. Within this model, the pK of the distal histidine is found to be 6.0 in the "open" configuration and 3.8 in the "closed" conformation. This corresponds to a 3 kcal/mol destabilization of the positively charged distal histidine within the hydrophobic pocket and suggests how protonation can lead to a larger population of the "open" conformation. At pH 7, the pocket is found to be "open" approximately 3% of the time. Further work, involving both IR and Raman measurements, allows the electron-nuclear coupling strengths of the various nu Fe-CO and nu C-O Raman modes to be determined. The slowly rebinding conformational state, corresponding to nu Fe-CO = 518 cm-1 (nu C-O = 1932 cm-1), displays unusually weak coupling of the Fe-CO mode to the Soret transition. Studies of the nu Fe-CO region as a function of temperature reveal that the equilibria between the conformational states are quenched in both the native and glycine mutant below the freezing point of the solvent. Unusual line narrowing of the nu Fe-CO modes at the phase transition is also observed in all samples studied. This line narrowing stands in marked contrast to the other heme Raman modes and suggests that Fe-CO librational motion and/or distal pocket vibrational (or conformational) excitations are involved in the line broadening at room temperature.     

Infrared spectroscopic studies of carbonyl horseradish peroxidases.

Infrared difference spectra, FeIIICO vs. FeIII of horseradish peroxidase isoenzymes A2 and C were recorded from 2000 to 1800 cm-1. Under alkaline conditions, pH 9, both isoenzymes exhibit two CO stretching bands, at 1938 and 1925 cm-1 for A2 and at 1933 and 1929 cm-1 for C. As the pH is lowered the low-frequency band for each isoenzyme decreases in intensity with a concommitant appearance and increase in intensity of a band at 1906 and 1905 cm-1 for the A2 and C isoenzymes, respectively. These changes conform to pK values of 6.7 for the A2 and 8.8 for the C isoenzymes of horseradish peroxidase. The interpretation of the infrared results was simplified by the observation that a linear relationship exists between the redox potential, Em7, for the FeIII/FeII system vs. the infrared CO stretching frequency, vCO, for cytochrome a3, hemoglobin, myoglobin, and cytochrome P-450 cam with substrate. This relationship suggests that the primary force altering vCO in these heme proteins is a variation in electron density at the heme iron and not direct protein interactions with the CO ligand. The horseradish peroxidase infrared bands in the 1930-cm-1 region correlate well with this relationship. The large deviation of the 1905-cm-1 band from the linear relationship and its dependence upon hydrogen ion concentration are consistent with horseradish peroxidase having a single CO binding site which can hold in two geometries, one of which contains an amino acid moiety capable of forming a hydrogen bond to the carbonyl oxygen.