The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

Modification of the heme distal side in myoglobin by cyanogen bromide. Heme environmental structures and ligand binding properties of the modified myoglobin

Met, deoxy, and CO forms of myoglobin (Mb) react with a stoichiometric amount of cyanogen bromide (BrCN) to cause substantial changes in the 1H NMR, optical absorption, and infrared spectra. These spectral changes were interpreted as arising from the substantial alterations in the heme environments, most probably due to the modification of the histidine residue at the heme distal side. It is also revealed that the modified Mb does not combine with some exogenous ligands such as CN-, CH3NH2, and O2, although it does with N-3 or CO. These unique ligand binding properties are also discussed with relevance to a role of the distal histidine in stabilizing the coordinated ligand through a hydrogen bond and to a steric constraint.     

Water penetration and binding to ferric myoglobin

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.     

Structure and ligand binding properties of leucine 29(B10) mutants of human myoglobin.

Site-specific mutants of human myoglobin (Mb) have been prepared, in which Leu29 (B10) is replaced by Ala(L29A) or Ile(L29I), in order to examine the influence of this highly conserved residue in the hydrophobic clusters of the heme distal site on the heme environmental structure and ligand binding properties of Mb. Structural characterizations of these recombinant Mbs are studied by electronic absorption, infrared (IR), one- and two-dimensional proton nuclear magnetic resonance spectroscopies, and ligand-binding kinetics by laser photolysis measurements under ambient and high pressures (up to 2000 bar). Multiple split carbon monoxide (CO) stretch bands in the IR spectra of mutant Mbs exhibit a relative decrease of the 1945 cm-1 band (approximately 50%) which is associated with an upright binding geometry of CO, accompanied by an increase of the tilted CO conformer at 1932 cm-1. On the basis of these results, replacement of Leu29(B10) by Ala or Ile appears to allow bound CO to rotate from a conformation pointing toward the beta meso carbon of the heme group to the one pointing toward the alpha meso carbon atom, presumably filling the space left by removal of the delta 2 carbon atom of Leu29(B10). These substitutions cause the rate constants for CO and O2 association to decrease almost 3-5-fold. Present results show that CO and O2 bindings to the heme iron of Mb are controlled by Leu29(B10) by influencing the structure of close vicinity of the heme and the geometry of iron-bound ligand. Further, mutant Mbs (Leu72(E15)----Ala and Leu104 (G5)----Ala) which have altered residues in another hydrophobic clusters around proximal and distal site are also examined.     

Dynamics of dioxygen and carbon monoxide binding to soybean leghemoglobin

The association of dioxygen and carbon monoxide to soybean leghemoglobin (Lb) has been studied by laser flash photolysis at temperatures from 10 to 320 K and times from 50 ns to 100 s. Infrared spectra of the bound and the photodissociated state were investigated between 10 and 20 K. The general features of the binding process in leghemoglobin are similar to the ones found in myoglobin. Below about 200 K, the photodissociated ligands stay in the heme pocket and rebinding is not exponential in time, implying a distributed enthalpy barrier between pocket and heme. At around 300 K, ligands migrate from the solvent through the protein to the heme pocket, and a steady state is set up between the ligands in the solvent and in the heme pocket. The association rate, lambda on, is mainly controlled by the final binding step at the heme, the bond formation with the heme iron. Differences between Lb and other heme proteins show up in the details of the various steps. The faster association rate in Lb compared to sperm whale myoglobin (Mb) is due to a faster bond formation. The migration from the solvent to the heme pocket is much faster in Lb than in Mb. The low-temperature binding (B----A) and the infrared spectra of CO in the bound state A and the photodissociated state B are essentially solvent-independent in Mb, but depend strongly on solvent in Lb. These features can be correlated with the x-ray structure.     

Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release

HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.     

Ligand binding to heme proteins: connection between dynamics and function

Ligand binding to heme proteins is studied by using flash photolysis over wide ranges in time (100 ns-1 ks) and temperature (10-320 K). Below about 200 K in 75% glycerol/water solvent, ligand rebinding occurs from the heme pocket and is nonexponential in time. The kinetics is explained by a distribution, g(H), of the enthalpic barrier of height H between the pocket and the bound state. Above 170 K rebinding slows markedly. Previously we interpreted the slowing as a "matrix process" resulting from the ligand entering the protein matrix before rebinding. Experiments on band III, an inhomogeneously broadened charge-transfer band near 760 nm (approximately 13,000 cm-1) in the photolyzed state (Mb*) of (carbonmonoxy)myoglobin (MbCO), force us to reinterpret the data. Kinetic hole-burning measurements on band III in Mb* establish a relation between the position of a homogeneous component of band III and the barrier H. Since band III is red-shifted by 116 cm-1 in Mb* compared with Mb, the relation implies that the barrier in relaxed Mb is 12 kJ/mol higher than in Mb*. The slowing of the rebinding kinetics above 170 K hence is caused by the relaxation Mb*----Mb, as suggested by Agmon and Hopfield [(1983) J. Chem. Phys. 79, 2042-2053]. This conclusion is supported by a fit to the rebinding data between 160 and 290 K which indicates that the entire distribution g(H) shifts. Above about 200 K, equilibrium fluctuations among conformational substates open pathways for the ligands through the protein matrix and also narrow the rate distribution. The protein relaxations and fluctuations are nonexponential in time and non-Arrhenius in temperature, suggesting a collective nature for these protein motions. The relaxation Mb*----Mb is essentially independent of the solvent viscosity, implying that this motion involves internal parts of the protein. The protein fluctuations responsible for the opening of the pathways, however, depend strongly on the solvent viscosity, suggesting that a large part of the protein participates. While the detailed studies concern MbCO, similar data have been obtained for MbO2 and CO binding to the beta chains of human hemoglobin and hemoglobin Zürich. The results show that protein dynamics is essential for protein function and that the association coefficient for binding from the solvent at physiological temperatures in all these heme proteins is governed by the barrier at the heme.     

Dynamics of ligand rebinding to unfolded MbCO by guanidine HCl

Femtosecond vibrational spectroscopy was used to probe a functionally important dynamics and residual structure of myoglobin unfolded by 4 M guanidine HCl. The spectra of the dissociated CO indicated that the residual structure of unfolded myoglobin (Mb) forms a few hydrophobic cavities that could accommodate the dissociated ligand. Geminate rebinding (GR) of CO to the unfolded Mb is three-orders-of-magnitude faster and more efficient than the native Mb but similar to a model heme in a viscous solvent, suggesting that the GR of CO to heme is accelerated by the longer retention of the dissociated ligand near the Fe atom by the poorly-structured protein matrix of the unfolded Mb or viscous solvent. The inefficient GR of CO in native Mb, while dissociated CO is trapped in the primary heme pocket located near the active binding site, indicates that the tertiary structure of the pocket in native Mb plays a functionally significant role.     

Exclusive CO binding to cytochrome oxidase

CO added to dithionite-reduced cytochrome oxidase pretreated with azide, cyanide, or fluoride yielded CO-ferrous heme a3 trapping the unliganded reduced heme. Ferrous heme a3 was either an equilibrium species initially present, or provided by dissociation of ligand-bound ferric heme a3 followed by the reduction with dithionite. In the latter case the ligand dissociation was rate-limiting for the CO compound formation. Pretreatment of the enzyme with the inhibitory ligands affected neither photodissociation and reassociation of the CO compound thus formed, nor reaction with dioxygen initiated by the flow-flash method to any significant degree. Only the cyanide treatment slightly decreased the rate of intramolecular electron transfer. These results indicate that no inhibitory ligand but CO remains in the vicinity of the heme a3-CuB center in the CO compound of cytochrome oxidase.     

Carbon monoxide binding to Rhodospirillum molischianum ferrocytochrome c'

Reversible carbon monoxide binding has been used to examine the structural and functional properties of reduced Rhodospirillum molischianum cytochrome c'. The symmetrical dimer is found to bind CO in a noncooperative manner, indicating that the heme sites function independently and with identical carbon monoxide affinity. The enthalpy change of binding CO (aqueous) to R. molischianum ferrocytochrome c' is determined to be -11 kcal/mol of CO, which is comparable to the heat of CO binding to other heme proteins. A Bohr effect is observed (0.31 +/- 0.04 proton released per mole of CO bound at pH 8), and a basic group is involved which changes its pK from 8.3 to 7.8 upon ligation. The histidine axial ligand to the heme iron is suggested to be the source of the Bohr effect. Increased CO affinities were observed at high pH or at neutral pH in the presence of phosphate. These solvent-induced changes in CO affinity do not appear to be caused by changes in quaternary structure but rather are more likely brought about by localized changes in the vicinity of the solvent-exposed heme face.     

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.     

An engineered heme–copper center in myoglobin: CO migration and binding

We have investigated CO migration and binding in Cu_BMb, a copper-binding myoglobin double mutant (L29H–F43H), by using Fourier transform infrared spectroscopy and flash photolysis over a wide temperature range. This mutant was originally engineered with the aim to mimic the catalytic site of heme–copper oxidases. Comparison of the wild-type protein Mb and Cu_BMb shows that the copper ion in the distal pocket gives rise to significant effects on ligand binding to the heme iron. In Mb and copper-free Cu_BMb, primary and secondary ligand docking sites are accessible upon photodissociation. In copper-bound Cu_BMb, ligands do not migrate to secondary docking sites but rather coordinate to the copper ion. Ligands entering the heme pocket from the outside normally would not be captured efficiently by the tight distal pocket housing the two additional large imidazole rings. Binding at the Cu ion, however, ensures efficient trapping in Cu_BMb. The Cu ion also restricts the motions of the His64 side chain, which is the entry/exit door for ligand movement into the active site, and this restriction results in enhanced geminate and slow bimolecular CO rebinding. These results support current mechanistic views of ligand binding in hemoglobins and the role of the Cu_B in the active of heme–copper oxidases. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

A kinetic description of ligand binding to sperm whale myoglobin

Nanosecond recombination time courses were measured by photolyzing O2, NO, CO, methyl, ethyl, n-propyl, n-butyl, and tert-butyl isocyanide complexes of sperm whale myoglobin with a 30-ns laser pulse at pH 7, 20 degrees C. Absorbance was measured both during and after the excitation pulse and as a function of laser light intensity. The results were analyzed quantitatively in terms of a three-step reaction scheme, MbX in equilibrium B in equilibrium C in equilibrium Mb + X, where Mb is myoglobin, B represents a geminate state in which the ligand is present in the distal pocket but not covalently bound to the iron atom, and C, a state in which the ligand is still embedded in the protein but further away from the heme group. The fitted rate parameters were required to be consistent with the observed overall quantum yield, Q, which had been measured independently using much longer (approximately 0.5 ms) xenon flash pulses. Three major conclusions were derived from these analyses. First, the overall quantum yield of the ligand complex is determined primarily by the competition between the rate of iron-ligand bond formation from the initial photoproduct, kB----MbX, and the rate of migration away from state B, kB----C. For example, kB----C approximately equal to 30-100 microseconds-1 for all three gaseous ligands, whereas both Q and kB----MbX vary over 3 orders of magnitude (i.e. NO, Q = 0.001, kB----MbX approximately equal to 16,000 microseconds-1; O2, Q = 0.1, kB----MbX approximately equal to 500 microseconds-1; CO, Q = 1.0, kB----MbX approximately equal to 2 microseconds-1). Second, for NO, O2, and the isonitriles, the rate-limiting step in the overall association reaction starting from ligand in solution is the formation of state B. The rate constant for this process varies from 2 X 10(7) M-1 s-1 for the gaseous ligands to 0.02-1.4 X 10(5) M-1 s-1 for the isonitriles. In contrast, the B to MbX transition is limiting for CO binding. Third, for all the ligands except CO, the overall rate of dissociation is limited significantly both by the rate of thermal bond disruption, kMbX----B, and the competition between geminate recombination and migration away from the distal pocket (i.e. kB----C/(kB----MbX + kB----C]. In the case of CO, the rate of bond disruption is equal to the observed dissociation rate constant.     

Kinetic study of CO and O2 binding to horse heart myoglobin reconstituted with synthetic hemes lacking methyl and vinyl side chains

Carbon monoxide- and oxygen-binding rates and affinities were measured for horse heart myoglobins reconstituted with synthetic hemes lacking peripheral methyl and vinyl groups. There is an apparent correlation between heme size and ligand specificity, i.e. larger m values (ratios of CO vs O2 association rates, l'/k') with smaller hemes. However, this correlation broke down with the most dealkylated heme. This is interpreted as resulting from protein conformational changes altering the steric crowdedness at the O2-binding site. Spectral properties and autoxidation rates also corroborate this view.     

Spectroscopic and photothermal study of myoglobin conformational changes in the presence of sodium dodecyl sulfate

Interactions between sodium dodecyl sulfate (SDS) and horse heart myoglobin (Mb) at surfactant concentrations below the critical micelle concentration have been studied using steady-state and transient absorption spectroscopies and photoacoustic calorimetry. SDS binding to Mb induces a heme transition from high-spin five-coordinate to low-spin six-coordinate in met- and deoxyMb, with the distal His residue likely to be the sixth ligand. The transition is complete at an SDS concentration of approximately 350 microM and approximately 700 microM for met- and deoxyMb, respectively. DeltaG(H(2)O) and m values determined from equilibrium SDS-induced unfolding curves indicate similar stability of met- and deoxyMb toward unfolding; however, the larger m value for the deoxyMb equilibrium intermediate indicates that its structure differs from that of metMb. Results from transient absorption spectroscopy show that CO rebinding to Fe(2+)-Mb in the presence of SDS is a biphasic process with the rate constant of the first process approximately 5.5 x 10(3) s(-1), whereas the second process displays a rate similar to that for CO rebinding to native Mb (k(obs) = 7.14 x 10(2) s(-1)) at 1 mM CO. Results of photoacoustic calorimetry show that CO dissociation from deoxyMb occurs more than 10 times faster in the presence of SDS than in native Mb. These data suggest that the heme binding pocket is more solvent-exposed in the SDS-induced equilibrium intermediate relative to native Mb, which is likely due to the electrostatic and hydrophobic interactions between surfactant molecules and the protein matrix.     

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).     

Can Gas Replace Protein Function? CO Abrogates the Oxidative Toxicity of Myoglobin

Outside their cellular environments, hemoglobin (Hb) and myoglobin (Mb) are known to wreak oxidative damage. Using haptoglobin (Hp) and hemopexin (Hx) the body defends itself against cell-free Hb, yet mechanisms of protection against oxidative harm from Mb are unclear. Mb may be implicated in oxidative damage both within the myocyte and in circulation following rhabdomyolysis. Data from the literature correlate rhabdomyolysis with the induction of Heme Oxygenase-1 (HO-1), suggesting that either the enzyme or its reaction products are involved in oxidative protection. We hypothesized that carbon monoxide (CO), a product, might attenuate Mb damage, especially since CO is a specific ligand for heme iron. Low density lipoprotein (LDL) was chosen as a substrate in circulation and myosin (My) as a myocyte component. Using oxidation targets, LDL and My, the study compared the antioxidant potential of CO in Mb-mediated oxidation with the antioxidant potential of Hp in Hb-mediated oxidation. The main cause of LDL oxidation by Hb was found to be hemin which readily transfers from Hb to LDL. Hp prevented heme transfer by sequestering hemin within the Hp-Hb complex. Hemin barely transferred from Mb to LDL, and oxidation appeared to stem from heme iron redox in the intact Mb. My underwent oxidative crosslinking by Mb both in air and under N₂. These reactions were fully arrested by CO. The data are interpreted to suit several circumstances, some physiological, such as high muscle activity, and some pathological, such as rhabdomyolysis, ischemia/reperfusion and skeletal muscle disuse atrophy. It appear that CO from HO-1 attenuates damage by temporarily binding to deoxy-Mb, until free oxygen exchanges with CO to restore the equilibrium.     

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 studies of CO and O2 binding to elephant myoglobin (distal His(E7)----Gln)

Carbon monoxide and dioxygen were employed as resonance Raman-visible ligands for probing the nature of the heme-binding site in elephant myoglobin, which has glutamine in the distal position (E7) instead of the usual histidine. The distal histidine (E7) residue has been thought to be responsible for weakening carbon monoxide binding to hemoproteins. It is of interest to see how the His(E7)----Gln replacement affects such parameters as nu(Fe-N epsilon), nu(Fe-CO), delta(Fe-C-O), nu(C-O), delta(Fe-O-O), and nu(O-O) vibrational frequencies and relative intensities. Elephant myoglobin has a CO affinity approximately 6 times higher than that for human/sperm whale myoglobin (Mb). If this enhanced affinity were solely due to the removal of some of the steric hindrance that normally tilts the CO off the heme axis, one would expect the nu(Fe-CO) frequency to decrease and the nu(C-O) frequency to increase relative to the corresponding values in sperm whale Mb. However, the opposite was found. In addition, strong enhancement of the Fe-C-O bending mode was observed. These results suggest that the Fe-C-O linkage remains distorted. In elephant Mb, new interactions resulting from the conformational change accompanying ligand binding may be responsible for the increased CO binding. Similar spectra were obtained for elephant and sperm whale oxymyoglobin. This suggests that the interactions of bound O2 are not markedly affected by the glutamine replacement.     

Ligand binding subsequent to CO photolysis of methionine-modified cytochrome c

In this report the kinetics of CO recombination to ferrocytochrome c in which Met80 has been oxidized to a sulfoxide are examined. Transient optical difference spectra suggest that the species formed immediately after photolysis contains a five-coordinate high spin heme. Single wavelength transient absorption data show triphasic kinetics with rate constants of (2.1+/-0.08)x10(4), (2.0+/-0.01)x10(3), and 57+/-0.01 s(-1). The data suggest a model for ligand recombination in which the methionine sulfoxide and CO compete for binding to the five-coordinate heme with rate constants of (2.1+/-0.08)x10(4) and (2.0+/-0.01)x10(3) s(-1), respectively. Carbon monoxide then binds to the population of cytochrome c containing the methionine sulfoxide with a rate constant of 57 s(-1). In addition, the slower than expected rate of methionine sulfoxide recombination (much smaller rate constant than expected for a ligand restricted to the distal heme pocket) is attributed to hydrogen bonding between the unbound methionine sulfoxide and Tyr(68).