Effect of phosphate on the kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains

The kinetics of assembly of oxygenated hemoglobin from isolated alpha and beta chains was investigated under various buffer conditions by use of a circular dichroism (CD) stopped-flow apparatus. The difference CD spectra of hemoglobin against its constituent chains were independent of the buffer conditions, while the time courses of the Soret CD after mixing equimolar amounts of the alpha and beta chains changed with the buffer conditions. The time courses were analyzed on the basis of a scheme which included a monomer-tetramer equilibrium of the beta chain (beta 4 in equilibrium 4 beta), dissociation of the beta 4 (beta 4 leads to 4 beta), and a second-order combination of alpha and beta monomers (alpha + beta leads to alpha beta). The analysis showed that buffer conditions affected the dissociation of the beta 4 rather than the monomer combination: The rate of the dissociation of the beta 4 accelerated with decreasing phosphate concentration, while the rate of the monomer combination was less sensitive to the phosphate concentration. This result indicates that the stability of the beta 4 depends on the phosphate concentration. It was furthermore suggested that the inorganic phosphate was bound to the beta 4 with an association constant of 133 M-1 and a Hill coefficient of 1.2.     

Reconstitution of myoglobin from apoprotein and heme, monitored by stopped-flow absorption, fluorescence and circular dichroism

The reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide was investigated with a stopped-flow apparatus by the use of five kinds of probes; (a) Soret absorption, (b) fluorescence quenching of tryptophan, (c) far-ultraviolet CD, (d) near-ultraviolet CD, and (e) Soret CD. After mixing of apomyoglobulin with equimolar amounts of hemin dicyanide, the Soret absorption band was shifted to longer wavelengths within 10 ms. The shifted band kept its shape for a few seconds, and then gradually shifted to shorter wavelengths. A rate constant of the slow reaction was 1.1 x 10(-2) s-1. Time courses of fluorescence quenching followed a second-order reaction with a rate constant of 9 x 10(7) M-1 s-1. Far-ultraviolet CD recovered to the level of native state within the response time of an apparatus (= 64 ms). Near-ultraviolet CD and Soret CD changed with first-order rate constants of 5-30 s-1 and 5 x 10(-3) s-1 respectively. On the basis of the kinetic results we propose the following reconstitution pathway of myoglobin. Apomyoglobin has essentially a highly folded structure similar to myoglobin, but there are some differences in the secondary structure between them. In the first step, heme enters the pocket-like site of apomyoglobin and interacts with surrounding hydrophobic residues in the pocket, and then the interaction may give a complete ordered structure to the protein. Second, the tertiary structure of the heme pocket is partly constructed. Third, the iron-proximal His bond occurs, followed by the attainment of the final conformation. This sequence of the events shows that the polypeptide chain is entirely folded before the completion of three-dimensional structure of the heme pocket. The reconstitution pathway is fairly different from that of the alpha subunit of hemoglobin reported by Leutzinger and Beychok [Proc. Natl Acad. Sci. USA (1981) 78, 780-784], which described how a drastic recovery in helicity was observed on the heme-binding, and that the recovery is introduced by the formation of the heme pocket structure. The difference in the results found for the alpha subunit and myoglobin suggests a difference in conformation: in apomyoglobin most of the helices are arranged and folded around a helix core to form a compact structure as a whole, while in apo-alpha subunit some helices are not folded around the helix core. Helix D, which is absent in the alpha subunit, may play an important role in folding of the helices.     

An investigation of human oxyhemoglobin beta tetramer dissociation using haptoglobin binding

Haptoglobin was used as a macromolecular probe to investigate the formation of human oxyhemoglobin beta chain dimers from tetramers in 0.1 M potassium phosphate buffer, 20 degrees C at pH 7 and pH 8. Monitoring of spectral changes upon mixing haptoglobin with beta heme chains (2.5 and 5 micromolar) revealed an overall decrease in absorbance accompanied by a shift of the Soret spectral peak from 415 to 417 nm. The magnitude of the absorbance decrease was proportional to the beta concentration; the time courses consistently yielded greater color at pH 8 than at pH 7. At pH 8, two exponential phases of 0.47 min-1 and 0.084 min-1 were seen whose rates remained invariant with concentration. In contrast, only one exponential process was evident at pH 7, yielding a first order rate constant of 0.21 min-1. We have spectrophotometrically followed the beta chain tetramer to dimer dissociation reaction, thus providing information about the contribution of this step to hemoglobin assembly.     

Electron transfer process in cytochrome bd-type ubiquinol oxidase from Escherichia coli revealed by pulse radiolysis

Cytochrome bd is a two-subunit ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and binds hemes b558, b595, and d as the redox metal centers. Taking advantage of spectroscopic properties of three hemes which exhibit distinct absorption peaks, we investigated electron transfer within the enzyme by the technique of pulse radiolysis. Reduction of the hemes in the air-oxidized, resting-state enzyme, where heme d exists in mainly an oxygenated form and partially an oxoferryl and a ferric low-spin forms, occurred in two phases. In the faster phase, radiolytically generated N-methylnicotinamide radicals simultaneously reduced the ferric hemes b558 and b595 with a second-order rate constant of 3 x 10(8) M-1 s-1, suggesting that a rapid equilibrium occurs for electron transfer between two b-type hemes long before 10 micros. In the slower phase, an intramolecular electron transfer from heme b to the oxoferryl and the ferric heme d occurred with the first-order rate constant of 4.2-5.6 x 10(2) s-1. In contrast, the oxygenated heme d did not exhibit significant spectral change. Reactions with the fully oxidized and hydrogen peroxide-treated forms demonstrated that the oxidation and/or ligation states of heme d do not affect the heme b reduction. The following intramolecular electron transfer transformed the ferric and oxoferryl forms of heme d to the ferrous and ferric forms, respectively, with the first-order rate constants of 3.4 x 10(3) and 5.9 x 10(2) s-1, respectively.     

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.     

Significance of beta116 His (G18) at alpha1beta1 contact sites for alphabeta assembly and autoxidation of hemoglobin

The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.     

Chicken liver sulfite oxidase. Kinetics of reduction by laser-photoreduced flavins and intramolecular electron transfer

Laser flash photolysis was used to study the reaction of photoproduced 5-deazariboflavin (dRFH.), lumiflavin (LFH.), and riboflavin (RFH.) semiquinone radicals with the redox centers of purified chicken liver sulfite oxidase. Kinetic studies of the native enzyme with dRFH. yielded a second-order rate constant of 4.0 X 10(8) M-1 s-1 for direct reduction of the heme and a first-order rate constant of 310 s-1 for intramolecular electron transfer from the Mo center to the heme. The reaction with LFH. gave a second-order rate constant of 2.9 X 10(7) M-1 s-1 for heme reduction. Reoxidation of the reduced heme due to intramolecular electron transfer to the Mo center gave a first-order rate constant of 155 s-1. The direction of intramolecular electron transfer using dRFH. and LFH. was independent of the buffer used for the experiment. The different first-order rate constants observed for intramolecular electron transfer using dRFH. and LFH. are proposed to result from chemical differences at the Mo site. Flash photolysis studies with cyanide-inactivated sulfite oxidase using dRFH. and LFH. resulted in second-order reduction of the heme center with rate constants identical with those obtained with the native enzyme, whereas the first-order intramolecular electron-transfer processes seen with the native enzyme were absent. The isolated heme peptide of sulfite oxidase gave only second-order kinetics upon laser photolysis and confirmed that the first-order processes observed with the native enzyme involve the Mo site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Esterification of the propionate groups promotes alpha/beta hemoglobin chain homogeneity of CN-hemin binding

This study examines the post-translational role of peripheral propionate groups in the incorporation of the Fe-protoporphryin IX heme into nascent alpha- and beta-globin chains. Human apohemoglobin (a heme-free alpha/beta dimer) in 0.05 M potassium phosphate buffer, pH 7, at 20 degrees C was titrated with either CN-protohemin (native heme with two peripheral propionate groups), or CN-dimethylester hemin (a modified heme with two methyl ester groups in place of the propionate groups). Soret spectrophotometric CN-hemin titrations confirmed that a spectral shift resulted upon binding of protohemin, but no spectral shift occurred upon binding the dimethylester derivative. Recent studies have correlated a Soret spectral shift with the preferential heme binding to the alpha subunit of apohemoglobin. The absence of a Soret wavelength shift (in conjunction with molecular modeling) presented here suggested that the modification of heme propionate groups prevented the formation of an alpha-heme/beta-globin intermediate, a requisite step in the normal assembly of functional hemoglobin.     

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

GroEL/GroES-dependent reconstitution of alpha2 beta2 tetramers of humanmitochondrial branched chain alpha-ketoacid decarboxylase. Obligatory interaction of chaperonins with an alpha beta dimeric intermediate

The decarboxylase component (E1) of the human mitochondrial branched chain alpha-ketoacid dehydrogenase multienzyme complex (approximately 4-5 x 10(3) kDa) is a thiamine pyrophosphate-dependent enzyme, comprising two 45.5-kDa alpha subunits and two 37.8-kDa beta subunits. In the present study, His6-tagged E1 alpha2 beta2 tetramers (171 kDa) denatured in 8 M urea were competently reconstituted in vitro at 23 degrees C with an absolute requirement for chaperonins GroEL/GroES and Mg-ATP. Unexpectedly, the kinetics for the recovery of E1 activity was very slow with a rate constant of 290 M-1 s-1. Renaturation of E1 with a similarly slow kinetics was also achieved using individual GroEL-alpha and GroEL-beta complexes as combined substrates. However, the beta subunit was markedly more prone to misfolding than the alpha in the absence of GroEL. The alpha subunit was released as soluble monomers from the GroEL-alpha complex alone in the presence of GroES and Mg-ATP. In contrast, the beta subunit discharged from the GroEL-beta complex readily rebound to GroEL when the alpha subunit was absent. Analysis of the assembly state showed that the His6-alpha and beta subunits released from corresponding GroEL-polypeptide complexes assembled into a highly structured but inactive 85.5-kDa alpha beta dimeric intermediate, which subsequently dimerized to produce the active alpha2 beta2 tetrameter. The purified alpha beta dimer isolated from Escherichia coli lysates was capable of binding to GroEL to produce a stable GroEL-alpha beta ternary complex. Incubation of this novel ternary complex with GroES and Mg-ATP resulted in recovery of E1 activity, which also followed slow kinetics with a rate constant of 138 M-1 s-1. Dimers were regenerated from the GroEL-alpha beta complex, but they needed to interact with GroEL/GroES again, thereby perpetuating the cycle until the conversion from dimers to tetramers was complete. Our study describes an obligatory role of chaperonins in priming the dimeric intermediate for subsequent tetrameric assembly, which is a slow step in the reconstitution of E1 alpha2 beta2 tetramers.     

A new ruthenium complex to study single-electron reduction of the pulsed O(H) state of detergent-solubilized cytochrome oxidase

The first step in the catalytic cycle of cytochrome oxidase, the one-electron reduction of the fully oxidized enzyme, was investigated using a new photoactive binuclear ruthenium complex, [Ru(bipyrazine)2]2(quaterpyridine), (Ru2Z). The aim of the work was to examine differences in the redox kinetics resulting from pulsing the oxidase (i.e., fully reducing the enzyme followed by reoxidation) just prior to photoreduction. Recent reports indicate transient changes in the redox behavior of the metal centers upon pulsing. The new photoreductant has a large quantum yield, allowing the kinetics data to be acquired in a single flash. The net charge of +4 on Ru2Z allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. The photoexcited state Ru(II*) of Ru2Z is reduced to Ru(I) by the sacrificial electron donor aniline, and Ru(I) then reduces CuA with yields up to 60%. A stopped-flow-flash technique was used to form the pulsed state of cytochrome oxidase (the "OH" state) from several sources (bovine heart mitochondria, Rhodobacter sphaeroides, and Paracoccus denitrificans). Upon mixing the fully reduced anaerobic enzyme with oxygenated buffer containing Ru2Z, the oxidized OH state was formed within 5 ms. Ru2Z was then excited with a laser flash to inject one electron into CuA. Electron transfer from CuA --> heme a --> heme a3/CuB was monitored by optical spectroscopy, and the results were compared with the enzyme that had not been pulsed to the OH state. Pulsing had a significant effect in the case of the bovine oxidase, but this was not observed with the bacterial oxidases. Electron transfer from CuA to heme a occurred with a rate constant of 20,000 s-1 with the bovine cytochrome oxidase, regardless of whether the enzyme had been pulsed. However, electron transfer from heme a to the heme a3/CuB center in the pulsed form was 63% complete and occurred with biphasic kinetics with rate constants of 750 s-1 and 110 s-1 and relative amplitudes of 25% and 75%. In contrast, one-electron injection into the nonpulsed O form of the bovine oxidase was only 30% complete and occurred with monophasic kinetics with a rate constant of 90 s-1. This is the first indication of a difference between the fast form of the bovine oxidase and the pulsed OH form. No reduction of heme a3 is observed, indicating that CuB is the initial electron acceptor in the one-electron reduced pulsed bovine oxidase.     

The heme-globin and dimerization equilibria of recombinant human hemoglobins carrying site-specific beta chains mutations

The heme-globin and dimer-tetramer equilibria of ferric recombinant human hemoglobins with site-specific beta chain mutations at the heme pocket or at either the a1beta1 or the alpha1beta2 interfaces have been determined. The heme pocket mutation V67T leads to a marked stabilization of the beta chain heme and does not affect the dimer-tetramer association constant, K2,4. In the C112 mutants, the intrinsic rate of beta chain heme loss with respect to recombinant HbA (HbA-wt) is significantly increased only in C112G with some heme released also from the alpha chains. Gel filtration experiments indicate that the K2,4 value is essentially unaltered in C112G and C112L, but is increased in C112V and decreased in C112N. Substitution of cysteine 93 with A or M leads to a slight decrease of the rate of beta chain heme release, whereas the obvserved K2,4 value is similar to that obtained for HbA-wt. Modifications in oxygen affinity were observed in all the mutant hemoglobins with the exception of V67T, C93A, and C112G. The data indicate that there is no correlation between tetramer stability, beta chain heme affinity, and hemoglobin functionality and therefore point to a separate regulation of these properties.     

A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1

Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2-0.5 s-1 at 24 degrees C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5-2 s-1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01-0.05 s-1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein.     

O2-mediated oxidation of hemopexin-heme(II)-NO

Hemopexin (HPX), serving as scavenger and transporter of toxic plasma heme, has been postulated to play a key role in the homeostasis of NO. Here, kinetics of HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO are reported. NO reacts reversibly with HPX-heme(II) yielding HPX-heme(II)-NO, according to the minimum reaction scheme: HPX-heme(II)+NO kon<-->koff HPX-heme(II)-NO values of kon, koff, and K (=kon/koff) are (6.3+/-0.3)x10(3)M-1s-1, (9.1+/-0.4)x10(-4)s-1, and (6.9+/-0.6)x10(6)M-1, respectively, at pH 7.0 and 10.0 degrees C. O2 reacts with HPX-heme(II)-NO yielding HPX-heme(III) and NO3-, by means of the ferric heme-bound peroxynitrite intermediate (HPX-heme(III)-N(O)OO), according to the minimum reaction scheme: HPX-heme(II)-NO+O2 hon<--> HPX-heme(III)-N(O)OO l-->HPX-heme(III)+NO3- the backward reaction rate is negligible. Values of hon and l are (2.4+/-0.3)x10(1)M-1s-1 and (1.4+/-0.2)x10(-3)s-1, respectively, at pH 7.0 and 10.0 degrees C. The decay of HPX-heme(III)-N(O)OO (i.e., l) is rate limiting. The HPX-heme(III)-N(O)OO intermediate has been characterized by optical absorption spectroscopy in the Soret region (lambdamax=409 nm and epsilon409=1.51x10(5)M-1cm-1). These results, representing the first kinetic evidence for HPX-heme(II) nitrosylation and O2-mediated oxidation of HPX-heme(II)-NO, might be predictive of transient (pseudo-enzymatic) function(s) of heme carriers.     

Chromatium vinosum cytochrome c-552. Reduction by photoreduced flavins and intramolecular electron transfer

Cytochrome c-552 from Chromatium vinosum is an unusual heme protein in that it contains two hemes and one flavin per molecule. To investigate whether intramolecular electron transfer occurs in this protein, we have studied its reduction by external photoreduced flavin by using pulsed-laser excitation. This approach allows us to measure reduction kinetics on the mirosecond time scale. Both fully reduced lumiflavin and lumiflavin semiquinone radical reduce cytochrome c-552 with second-order rate constants of approximately 1.4 x 10(6) M-1s-1 and 1.9 x 10(8) M-1 s-1, respectively. Kinetic and spectral data and the results of similar studies with riboflavin indicate that both the flavin and heme moieties of cytochrome c-552 are reduced simultaneously on a millisecond time scale, with the transient formation of a protein-bound flavin anion radical. This is suggested to be due to rapid intramolecular electron transfer. Further, steric restrictions play an important role in the reduction reaction. Studies were conducted on the redox processes following photolysis of CO-ferrocytochrome c-552 in which the flavin was partly oxidized to resolve the kinetics of electron transfer between the heme and flavin of cytochrome c-552. Based on these results, we conclude that intramolecular electron transfer from ferrous heme to oxidized flavin occurs with a first-order rate constant of greater than 1.4 x 10(6) s-1.     

Optical detection of a red-shifted Michaelis complex in the reaction of HCN with ferromyeloperoxidase. Evidence for conformational stabilization of high spin iron (II).

The reaction of HCN with ferromyeloperoxidase involves the sequential formation of two monocyanide complexes. The first complex, which forms immediately on mixing, is characterized by a red shift in the Soret band of the ferroperoxidase, and a dissociation constant (measured as a Michaelis constant) of 0.67 mM. The second complex arises from the first via a first order process, whose maximal rate is 0.095 s-1 at 25 degrees C, pH 7.0. This more stable complex is characterized by a blue shift in the Soret and alpha bands and by an overall dissociation constant in the region of 4.5 microM. This gives a free energy difference between the two complexes of around 3.0 kcal mol-1 and a difference in optical absorption of 15 nm (Soret). The measured Arrhenius activation energy for the conversion of the high energy, long wavelength complex to the low energy, short wavelength complex is 16.3 kcal mol-1. A larger blue shift is observed on protein denaturation (34 nm), after which the two-step binding reaction is not observed. This, and the magnitude of the activation energy in the spontaneous complex interconversion process, shows that the latter is a conformational process. In addition, it can be concluded that the unknown structural feature of the heme site which is responsible for the anomalous red shift in the optical spectrum of native ferromyeloperoxidase, is also the link between the ligand state of the iron and the protein conformation.     

Binding reaction of hemin to globin

Binding of hemin to globin was studied in the presence of 25 mM caffeine by measuring CD and optical absorption changes in the Soret region. CD and optical absorption spectra after mixing equimolar amounts of hemin and globin were the same as those of ferric hemoglobin. In contrast, addition of excess globin to hemin formed a complex that was distinguishable from ferric hemoglobin in terms of the CD and optical absorption spectra. By comparing the spectra of the complex with those of various hemoglobin derivatives, it was concluded that the complex was globin which carried a hemin exclusively on the alpha chain. This means that the alpha chain of the globin molecule has a greater affinity for hemin than the beta chain, as observed by other investigators using hemin-cyanide. The rate of binding of hemin to globin was estimated by the use of CD and optical absorption stopped-flow apparatus. The rate of hemin binding to the alpha chain of globin was obtained by mixing hemin and excess globin, and that to the beta chain was obtained by mixing equimolar concentrations of hemin and globin. The results showed that hemin was bound to the alpha chain in the globin molecule to form a transient intermediate, followed by its transformation into another intermediate, the transformation was the rate-limiting step, and the beta chain in the globin molecule had a greater affinity for hemin after hemin binding to the alpha chain than before.     

The thermal denaturation of human oxyhaemoglobins A, A2, C and S

1. The time-courses of thermal denaturation of human oxyhaemoglobins A, A(2), C and S at 45 degrees C were studied by following the increase in protein fluorescence. Haemoglobins S and C were less stable than haemoglobin A, whereas haemoglobin A(2) was considerably more stable. 2. The time-courses of denaturation did not follow first-order kinetics and could be fitted most simply to a co-operative scheme in which the partial denaturation of the alpha chain preceded that of the beta chain. 3. The denaturation of these haemoglobins was studied as a function of temperature by using optical rotatory dispersion. Haemoglobin A(2) was again more stable than the others. The addition of small quantities of haemoglobin A(2) had a disproportionate effect on the stability of haemoglobin C. 4. The thermodynamic parameters of the denaturation process were calculated.     

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.

The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.