A stopped-flow study of the cupric ion oxidation of adult-human haemoglobin.

The addition of excess Cu2+ to adult human haemoglobin leads to the production of alpha 2(2+) beta 2(3+), in both the oxy and deoxy forms of the protein. Stopped-flow studies of the oxidation process yields apparent second-order rate constants of 196M-1 X S-1 and 41M-1 X S-1 for the deoxy and oxy forms respectively. The rate of the deoxy-form oxidation is linearly dependent on [Cu2+], whereas that of the oxy form is rate-limited above 2 mM to 0.11 S-1. Arrhenius activation energies of the two processes are almost identical at 91 kJ X mol-1, as are the activation enthalpies of 89 kJ X mol-1. The activation entropies show small differences, being 31 entropy units and 48 entropy units for the oxy and deoxy forms respectively. ATP and glycerate 2,3-bisphosphate at saturating concentrations do not affect the rate of oxidation of the oxy form, but halve the rate found for the deoxy form. These data are discussed in terms of the previously proposed mechanism of oxidation in which slow Cu2+ binding is followed by rapid electron transfer.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

pH Dependence of oxy and deoxy cobalt-substituted leghemoglobin from soybean

In leghemoglobin a, which is the major hemoglobin component in soybean root nodules, the haem iron has been replaced by cobalt. The electron spin resonance (ESR) of frozen solutions of the cobalt-substituted leghemoglobin has been studied at 77 K in the deoxy and oxy forms respectively. Both ligation states exhibit rhombic g tensors. The hyperfine constants of ⁵⁹Co, ¹⁴N-imidazole (residue of the proximal histidine) and ¹⁴N-pyrroles are determined for the three principal directions of the g tensor. Both, the oxy and the deoxy state exhibit pH-dependent changes of the hyperfine structures. For oxy cobalt leghemoglobin a quantitative analysis of the pH titration and of the ESR parameters of the low and high-pH forms respectively are performed. The interconversion of the low and the high-pH forms is controlled by a proton-dissociating group with pK=6.4 which is most probably the distal histidine. g tensors and hyperfine constants are compared with those described for oxy cobalt myoglobin crystal spectra [34] allowing assignments of the low and high-pH species of leghemoglobin to stereoelectronic structures with non-equivalent and equivalent dioxygen atoms respectively. Hydrogen-bonding of the distal histidine with dioxygen favours the structure with equivalent oxygen atoms. The pH dependence of the deoxy form is interpreted as interaction of the proximal imidazole with the central cobalt atom.     

Hemoglobin-water interactions in normal and sickle erythrocytes by proton magnetic resonance T1ϱ measurements

The dependence of the water proton magnetic resonance spin-lattice relaxation rate (T_1?^?1) in the rotating frame on the strength of the spin-locking (H₁) field has been investigated for packed oxy and deoxy normal and sickle erythrocytes at temperatures from 9 to 40 °C. The T_1?^?1 of oxy or deoxy normal erythrocytes shows no dependence on H₁ up to ~7 G at any temperature studied. On the other hand, T_1?^?1 decreases from about 40 s^?1 to 15 s^?1 (H₁ from 0 to ~7 G) for deoxygenated packed sickle cells at 40 °C. The magnitude of this variation of T_1?^?1 with H₁ decreases with decreasing temperature. Oxy packed sickle cells also show a dependence of T_1?^?1 on H₁ but the magnitude is <10% of that of the deoxygenated samples. These results suggest that water proton T_1?^?1 measurements are a sensitive probe of hemoglobin S polymerization and provide a novel technique for the study of slow water motions in these systems. The T_1?^?1 results are compared with low frequency T₁^?1 results of other investigators on hemoglobin S solutions. Analysis of the data suggests that water proton motions with correlation times of the order of 10^?5 s are present in the deoxygenated sickle cell samples at temperatures above 10 °C.     

Differential pathways in oxy and deoxy HbC aggregation/crystallization

CC individuals, homozygous for the expression of beta(C)-globin, and SC individuals expressing both beta(S) and beta(C)-globins, are known to form intraerythrocytic oxy hemoglobin tetragonal crystals with pathophysiologies specific to the phenotype. To date, the question remains as to why HbC forms in vivo crystals in the oxy state and not in the deoxy state. Our first approach is to study HbC crystallization in vitro, under non-physiological conditions. We present here a comparison of deoxy and oxy HbC crystal formation induced under conditions of concentrated phosphate buffer (2g% Hb, 1. 8M potassium phosphate buffer) and viewed by differential interference contrast microscopy. Oxy HbC formed isotropic amorphous aggregates with subsequent tetragonal crystal formation. Also observed, but less numerous, were twisted, macro-ribbons that appeared to evolve into crystals. Deoxy HbC also formed aggregates and twisted macro-ribbon forms similar to those seen in the oxy liganded state. However, in contrast to oxy HbC, deoxy HbC favored the formation of a greater morphologic variety of aggregates including polymeric unbranched fibers in radial arrays with dense centers, with infrequent crystal formation in close spatial relation to both the radial arrays and macroribbons. Unlike the oxy (R-state) tetragonal crystal, deoxy HbC formed flat, hexagonal crystals. These results suggest: (1) the Lys substitution at beta6 evokes a crystallization process dependent upon ligand state conformation [i. e., the R (oxy) or T (deoxy) allosteric conformation]; and (2) the oxy ligand state is thermodynamically driven to a limited number of aggregation pathways with a high propensity to form the tetragonal crystal structure. This is in contrast to the deoxy form of HbC that energetically equally favors multiple pathways of aggregation, not all of which might culminate in crystal formation.     

Evaluation of the water environments in deoxygenated sickle cells by longitudinal and transverse water proton relaxation rates

The longitudinal and transverse water proton relaxation rates of oxygenated and deoxygenated erythrocytes from both normal adults and individuals with sickle cell disease were measured as a function of temperature at two different frequencies. The simplest model which fits all of the data consists of three different environments for water molecules. The majority of the water (98%) has a correlation time indistinguishable from bulk water (3 × 10^?11 sec). Secondly, there is a small amount of water (1.3–1.5%) present which has a correlation time of 2–4 × 10 ^?9 sec and is apparently independent of the erythrocyte sample studied. Presumably this water is the hydration sphere around the hemoglobin molecules and its correlation time is significantly slower than bulk water. The third environment contains approximately 0.2% of the water present and has a correlation time≥ 10^?7 sec. This third environment is considered tightly bound to the hemoglobin because the water proton correlation time is very similar to the expected rotational correlation time for the hemoglobin molecules. The value of the transverse relaxation rate, f_b(T_2b)^?1, for the tightly bound water fraction decreases in oxy ($\text{S}\text{S}$), deoxy ($\text{A}\text{A}$), and oxy ($\text{A}\text{A}$) erythrocyte samples as the temperature is increased as expected for a rotational correlation time process. In dramatic contrast,f_b (T_2b)^?1 increases almost linearly as the temperature is increased over the whole 4 ° to 37 °C temperature range in samples of deoxy ($\text{S}\text{S}$) erythrocytes. The observation suggests a continual increase in the formation of deoxyhemoglobulin S polymers rather than a sudden transition from a homogeneous solution of deoxyhemoglobin S molecules to a solid gel.     

Computationally accessible method for estimating free energy changes resulting from site-specific mutations of biomolecules: systematic model building and structural/hydropathic analysis of deoxy and oxy hemoglobins

A practical computational method for the molecular modeling of free-energy changes associated with protein mutations is reported. The de novo generation, optimization, and thermodynamic analysis of a wide variety of deoxy and oxy hemoglobin mutants are described in detail. Hemoglobin is shown to be an ideal candidate protein for study because both the native deoxy and oxy states have been crystallographically determined, and a large and diverse population of its mutants has been thermodynamically characterized. Noncovalent interactions for all computationally generated hemoglobin mutants are quantitatively examined with the molecular modeling program HINT (Hydropathic INTeractions). HINT scores all biomolecular noncovalent interactions, including hydrogen bonding, acid-base, hydrophobic-hydrophobic, acid-acid, base-base, and hydrophobic-polar, to generate dimer-dimer interface "scores" that are translated into free-energy estimates. Analysis of 23 hemoglobin mutants, in both deoxy and oxy states, indicates that the effects of mutant residues on structurally bound waters (and visa versa) are important for generating accurate free-energy estimates. For several mutants, the addition/elimination of structural waters is key to understanding the thermodynamic consequences of residue mutation. Good agreement is found between calculated and experimental data for deoxy hemoglobin mutants (r = 0.79, slope = 0.78, standard error = 1.4 kcal mol(-1), n = 23). Less accurate estimates were initially obtained for oxy hemoglobin mutants (r = 0.48, slope = 0.47, standard error = 1.4 kcal mol(-1), n = 23). However, the elimination of three outliers from this data set results in a better correlation of r = 0.87 (slope = 0.72, standard error = 0.75, n = 20). These three mutations may significantly perturb the hemoglobin quaternary structure beyond the scope of our structural optimization procedure. The method described is also useful in the examination of residue ionization states in protein structures. Specifically, we find an acidic residue within the native deoxy hemoglobin dimer-dimer interface that may be protonated at physiological pH. The final analysis is a model design of novel hemoglobin mutants that modify cooperative free energy (deltaGc)--the energy barrier between the allosteric transition from deoxy to oxy hemoglobin.     

A comparative study on the structural differences of primate hemoglobins by spin labeling technique

The hemoglobins of human and five non-human primates were spin-labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide, and the ESR spectra of their deoxy, oxy, and carbonmonoxy forms were measured. The analyses of the spectra indicated that the local protein conformation in the vicinity of the spin-labeled cysteine residue at position 93(F9) in the beta-chain is slightly but significantly different among species, and that each hemoglobin shows a similar change in conformation upon conversion from the oxy form to the carbonmonoxy one except for human hemoglobin. Human hemoglobin was suggested to undergo a significantly different conformational change upon this conversion, indicating that it has unique characteristics among the primate hemoglobins.     

Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering

The influence of quaternary structure on the low frequency molecular vibrations of the haem within deoxyhaemoglobin (deoxy Hb) and Oxyhaemoglobin (oxy Hb) was studied by resonance Raman scattering. The FeO₂ stretching frequency was essentially identical between the high affinity (R) state (Hb A) and low affinity (T) state (Hb Kansas and Hb M Milwaukee with inositol hexaphosphate). However in deoxy Hb, only one of the polarized lines showed an appreciable frequency shift upon switch of quaternary structure, i.e. 215 to 218 cm^?1 for the T state (Hb A, des-His(146β) Hb, and des-Arg(141α) Hb (pH 6.5)) and 220 to 221 cm^?1 for the R state (des-Arg(141α) Hb (pH 9.0), des-His(146β)-Arg(141α) Hb and NES des-Arg(141α) Hb). Based on the observed ⁵⁴Fe isotopic frequency shift of the corresponding Raman lines of deoxy Hb A (214 → 217 cm^?1), of deoxy NES des-Arg Hb (220 → 223 cm^?1), of the protoporphyrinato-Fe(II)-(2-methylimidazole) complex in the ferrous high spin state (207 → 211 cm^?1) and of deoxymyoglobin (220 → 222 cm^?1) (Kitagawa et al., 1979), and on substitution of perdeuterated for protonated 2-methylimidazole in the deoxygenated picket fence complex (T_pivPP)Fe²⁺ (2-MeIm) (209 → 206 cm^?1), and on the results of normal co-ordinates calculation carried out previously, we proposed that the 216 cm^?1 line of deoxy Hb is associated primarily with the FeN_ε(HisF8) stretching mode and accordingly that the FeN_ε(HisF8) bond is stretched in the T state due to a strain exerted by globin.     

Heat stabilization dependence on redox state of cytochrome cd1 oxidase from Pseudomonas aeruginosa

The irreversible thermal denaturation of cytochrome cd₁ oxidase from $\text{P.}\text{aeruginosa}$ as a function of the oxidation-reduction states of its hemes was observed with a differential scanning calorimeter. Upon full reduction of the four hemes, the apparent denaturation temperature decreases by about 10° and the denaturation enthalpy decreases slightly: oxidized, 5.9 cal/gm; reduced, 5.4 cal/gm. At pH 7.5, the first order rate constants for denaturation at 90°C are: reduced, 33 × 10^?3s^?1; oxidized, 3 × 10^?3s^?1. Thus, oxidation of the hemes reuults in heat stabilization of the cytochrome oxidase. The activation energy for denaturation of fully reduced oxidase, 53 kcal/mol, is less than that for fully oxidized protein (73 kcal/mol).     

Inhibition of human hemoglobin autoxidation by sodium n-dodecyl sulphate

The effect of sodium n-dodecyl sulphate (SDS) on hemoglobin autoxidation was studied in the presence of a 100 mM phosphate buffer (pH 7.0) by different methods. These included spectrophotometry, fluorescence technique, cyclic voltametry, differential scanning calorimetry, and densitometry. Spectroscopic studies showed that SDS concentrations up to 1 mM increased deoxy-, decreases oxy-, and had no significant effect on the met- conformation of hemoglobin. Therefore, a SDS concentration up to 1 mM increased the deoxy form of hemoglobin as the folded, compact state and decreases the oxy conformation. The turbidity measurements and differential scanning calorimetry techniques indicated a more stable conformation for hemoglobin in the presence of SDS up to 1 mM. Electrochemical studies also confirmed a more difficult oxidation under these conditions. The induction of the deoxy form in the presence of SDS was confirmed by densitometry techniques. The compact structure of deoxyhemoglobin blocks the formation of met-conformation in low SDS concentrations.     

Studies on cobalt myoglobins and hemoglobins, XIII. A consequence of the occurrence of glutamine at the E7 (58) site of alpha subunits in opossum hemoglobin

To investigate the mode of interactions between heme metal, bound oxygen and the distal residue at the E7 site, we have measured accurate oxygen equilibrium curves, oxygen binding relaxations following temperature-jump, and electron paramagnetic resonance spectra of natural and cobalt-substituted opossum hemoglobin, which has glutamine and histidine at the E7 site of the α chain and the β chain, respectively, and compared them with those of natural and cobalt-substituted human hemoglobin, which has histidine at the E7 site of both the α and β chains.Natural opossum hemoglobin has a lower oxygen affinity, slightly smaller and pH-dependent co-operativity, a somewhat greater Bohr effect, and a smaller effect of organic phosphates such as 2,3-diphosphoglycerate and inositol hexaphosphate on oxygen affinity as compared to natural human hemoglobin. Upon substitution of cobalt for iron, these oxygenation characteristics of opossum hemoglobin relative to those of human hemoglobin were preserved well. The behavior of the intrinsic oxygen association constants pertaining to the four oxygenation steps (i.e. the Adair constants) upon addition of the organic phosphates or pH changes indicates that the allosteric equilibrium in opossum hemoglobin is biased towards the T state as compared with that in human hemoglobin, and that the oxygen affinity of the R structure is lower for opossum hemoglobin than for human hemoglobin. The temperature-jump kinetic data indicate that the lower oxygen affinity of opossum cobalt-hemoglobin in comparison with that of human cobalt-hemoglobin can be ascribed to a decreased oxygen association rate constant. The electron paramagnetic resonance experiments on oxy and deoxy opossum and human cobalt-hemoglobins in buffered H₂O and ²H₂O, including their photolysed products at a low temperature, provided the following information. The cobaltous ion of the α subunits of deoxy opossum cobalt-hemoglobin is in an environment that is similar to that for cobaltous ions of deoxy human cobalt-hemoglobin in the T state. The hydrogen bond between the bound oxygen and the residue at E7, which has been shown to exist in oxy human cobalt-hemoglobin and oxy sperm whale cobalt-myoglobin, is absent or, at least, significantly altered in the α subunits of oxy opossum cobalt-hemoglobin, probably resulting in a lower oxygen affinity. Interference by isoleucine at E11α with an oxygen molecule is suggested as an explanation for the lowered affinity of opossum iron-hemoglobin. However, no straightforward structural explanation is available for the lower oxygen affinity of the R structure and the allosteric equilibrium biased towards the T state in opossum iron-hemoglobin.     

Hydrogen-tritium exchange survey of allosteric effects in hemoglobin

The oxy and deoxy forms of hemoglobin display major differences in H-exchange behavior. Hydrogen-tritium exchange experiments on hemoglobin were performed in the low-resolution mode to observe the dependence of these differences on pH (Bohr effect), organic phosphates, and salt. Unlike a prior report, increasing pH was found to decrease the oxy-deoxy difference monotonically, in general accordance with the alkaline Bohr effect. A prior report that the H-exchange difference between oxy- and deoxyhemoglobin vanishes at pH 9, and thus appears to reflect the Bohr effect alone, was found to be due to the borate buffer used, which at high pH tends to abolish the oxy-deoxy difference in a limited region of the H-exchange curve. Effects on hemoglobin H exchange due to organic phosphates parallel the differential binding of these agents (inositol hexaphosphate more than diphosphoglycerate, deoxy more than oxy, at low pH more than at high pH). Added salt slows H exchange of deoxyhemoglobin and has no effect on the oxy form. These results display the sensitivity of simple H-exchange measurements for finding and characterizing effects on structure and dynamics that may occur anywhere in the protein and help to define conditions for higher resolution approaches that can localize the changes observed.     

Formation of a steady state in the radiolysis of ferrimyoglobin in aqueous solution

The interaction of radiation-generated · OH radicals with ferrimyoglobin in deaerated aqueous solution at neutral pH has been quantitatively studied. Changes in the visible absorption spectrum have been analyzed on the basis of composition changes of the ferri, deoxy, and ferriperoxide forms of the metalloprotein. A postirradiation thermal process must be considered in order to evaluate the radical-induced composition changes. Initially, ·OH induces reduction of ferrimyoglobin to the deoxy form with a G value (molecular yield/100 eV of absorbed energy) in the zero-dose limit of 1.4 (±0.2). Radiation-generated H₂O₂ reacts with the ferrimyoglobin substrate to produce ferrimyoglobin peroxide with a G value of 0.7 (±0.1) in the zero-dose limit. At doses of >1 krad μm^?1 of myoglobin present, the composition of the three myoglobin derivatives reaches a radiolysis steady state. In this moderate-dose plateau region, this composition is 44% ferri, 18% deoxy, and 38% ferri peroxide. The · OH-induced hemoprotein radicals that do not initiate 1-eq redox conversions undergo reactions that generate dimer and other globin-modified material.     

A comparison of the binding of imidazole to valence hybrid and methemoglobin: an assignment of individual heme reactivity

The ferric hemes of valence hybrid hemoglobins combine with imidazole in a manner analogous with the hemes of methemoglobin. Equilibrium studies show that imidazole binding to methemoglobin is minimally described by the sum of two independent processes (K₁ = 200 M^?1 and K₂ = 37 M^?1), both of which contribute equally to the observed difference spectrum. Using valance hybrid hemoglobins, which show single binding processes under similar conditions, it is possible to identify the high affinity sites in methemoglobin with the α chains and the low affinity sites with the β chains.Kinetic studies show that the valance hybrid hemoglobins react in a single exponential fashion with imidazole in contrast with methemoglobin which shows a biphasic reaction (k₁ = 85 M^?1 sec^?1k₂ = 25 M^?1 sec^?1). A comparison of the rates of reaction of the hybrids allows the assignment of the fast phase in methemoglobin to the β chains and the slow phase to the α chains.The heterogeneity of the imidazole reaction with methemoglobin occurs over the pH range 5.5–9.5 within which two ionization processes are discernable at pH 6.9 and 7.5.     

Calculation of the energy difference between the quaternary structures of deoxy- and oxyhemoglobin.

The energy difference between the quaternary structures of deoxy- and oxyhemoglobin is evaluated on the basis of the atomic coordinates determined by X-ray diffraction analysis. Calculation of the van der Waals interaction between subunits shows that in a hemoglobin molecule as a whole, the interaction is more attractive in the oxy form than in the deoxy form by about 8 kcal/mol, and that in each pair of two subunits except the pair alpha1alpha2, the interaction energy varies by about 15 kcal/mol. The electrostatic interactions originating in the partial charges on all constituent atoms of hemoglobin and in the polar residues on the surface of hemoglobin make only a small contribution to the energy difference between the quaternary structures of deoxy- and oxyhemoglobin. Thus, the contribution of the clusters of the polar residues in the internal cavity between like subunits and also of the freedom of rotation of the C-terminal of each subunit in oxyhemoglobin may be important energetically in the transition from deoxy to oxy quaternary structure. In this point, the present calculation supports Perutz' model, but suggests necessity of further investigations on the transitional characteristics of the quaternary structure in the intermediate steps of oxygenation. The discussion on the transitional characteristics is given in the last section.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

Low temperature photodissociation studies of ferrous hemoglobin and myoglobin complexes by Mössbauer spectroscopy

⁵⁷Fe-enriched complexes of hemoglobin and myoglobin with CO and O₂ were photodissociated at 4.2°K, and the resulting spectra were compared with those of the deoxy forms. Differences in both quadrupole splitting and isomer shift were noted for each protein, the photoproducts having smaller isomer shift and larger quadrupole splitting than the deoxy forms. The photoproducts of HbCO and HbCO₂ had narrow absorption lines, indicating a well-defined iron environment. The corresponding myoglobin species had broader absorption lines, as did both deoxy forms. The weak absorption lines of photodissociated NO complexes appeared to be wide, possibly indicating magnetic interaction with the unpaired electron of the nearby NO.