Geminate combination of oxygen with iron-cobalt hybrid hemoglobins

Photodissociation of oxygen from the ferrous subunits of hybrid hemoglobins in which the heme of either the alpha or the beta chain has been replaced by cobalt protoporphyrin IX shows large differences between the subunits. With a 25-ns light pulse, the apparent quantum yield at the end of the flash is greater for the beta-iron hybrid than for the alpha-iron hybrid. With the beta-iron hybrid, the yield is greater when solution conditions favor the T-state. After the flash, a part of the oxygen which has been dissociated recombines with a half-time of the order of tens of nanoseconds. The proportion is greatest in the R-state at low temperature and least in the T-state. With the alpha-iron hybrid, oxygen is much less readily removed, and the rapid recombination is slight or absent. It is seen most clearly at low temperatures in conditions which favor the T-state. The long term (greater than 100 ns) effect is that oxygen is much more readily removed from the beta-iron hybrid in the T-state than under any other condition. Analogous flash experiments performed with human hemoglobin A may be closely simulated by superposition of the results obtained with the two hybrid hemoglobins under the same conditions. Isolated human alpha and beta--SH chains show differences similar to, but less marked than, those of the iron-cobalt hybrids.     

Geminate reactions of oxygen and nitric oxide with the alpha and beta subunits of Fe-Co hybrid hemoglobins

The alpha and beta subunits in Fe-Co hybrid hemoglobins differ in their rapid reactions with dioxygen and nitric oxide after dissociation by a 25-ns photoflash. The alpha subunits show little recombination on a scale of tens of nanoseconds, whereas the beta subunits show extensive recombination on this time sale. The alpha-beta difference is more marked with Fe than with Co and greater with dioxygen as ligand than with nitric oxide, but is clearly evident in all combinations of ligand and metal. Addition of inositol hexaphosphate slows ligand binding and reduces the proportion of rapid recombination of dioxygen and nitric oxide to beta-Fe subunits. The behavior of alpha-Fe subunits is unaffected by this compound. These results permit the beta subunit to be identified as the T-state species which equilibrates rapidly with oxygen in the T-state, i.e. the reverse of the identification suggested on structural grounds.     

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k_off) is reported. In the absence of drugs, the value of k_off is (1.3 ± 0.2) × 10⁻⁴ s⁻¹. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k_off value increases to (8.6 ± 0.9) × 10⁻⁴ s⁻¹. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 ± 0.2) × 10⁻³ M and (6.2 ± 0.7) × 10⁻⁵ M, respectively) were determined. The increase of k_off values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow’s site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.     

Parameters controlling the kinetics of ferric and ferrous hemeproteins reduction by hydrated electrons

To clarify the processes of hemeproteins reduction, three classes of these proteins (ferric, ferrous and desFe) were reduced by hydrated electrons generated by pulse radiolysis. Spectral and kinetic investigations were made on alpha hemoglobin chain and myoglobin. Human alpha chain has been chosen to avoid all ferric contaminations and horse ferric myoglobin to eliminate all ferrous protein fractions. We have successively studied the influences of: the iron presence, its oxidation state (II and III), the protein charge and the iron-ligand nature (H2O, OH-, N3- and CN-). For alpha human hemoglobin chain without metallic ion or with ferrous iron, the reduction rates are the same: 1.1 +/- 0.2.10(10) M-1.s-1. In the case of horse ferric myoglobin, the reduction rates depend principally on the protein charge (from pH 6.3 to pH 9.5, the reduction rate of Mb(FeIII)N3- decreases from 2.5 +/- 0.5.10(10) M-1.s-1 to 1.2 +/- 0.2.10(10) M-1.s-1) and are also modulated by the equilibrium constant of the hemeprotein-ligand association (1.2 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)N3- and 0.8 +/- 0.2.10(10) M-1.s-1 for Mb(FeIII)CN-, at pH 9.8).     

Manganese peroxidase from the lignin-degrading basidiomycete Phanerochaete chrysosporium. Transient state kinetics and reaction mechanism

Stopped-flow techniques were used to investigate the kinetics of the formation of manganese peroxidase compound I (MnPI) and of the reactions of MnPI and manganese peroxidase compound II (MnPII) with p-cresol and MnII. All of the rate data were obtained from single turnover experiments under pseudo-first order conditions. In the presence of H2O2 the formation of MnPI is independent of pH over the range 3.12-8.29 with a second-order rate constant of (2.0 +/- 0.1) x 10(6) M-1 s-1. The activation energy for MnPI formation is 20 kJ mol-1. MnPI formation also occurs with organic peroxides such as peracetic acid, m-chloroperoxybenzoic acid, and p-nitroperoxybenzoic acid with second-order rate constants of 9.7 x 10(5), 9.5 x 10(4), and 5.9 x 10(4) M-1 s-1, respectively. The reactions of MnPI and MnPII with p-cresol strictly obeyed second-order kinetics. The second-order rate constant for the reaction of MnPII with p-cresol is extremely low, (9.5 +/- 0.5) M-1 s-1. Kinetic analysis of the reaction of MnII with MnPI and MnPII showed a binding interaction with the oxidized enzymes which led to saturation kinetics. The first-order dissociation rate constants for the reaction of MnII with MnPI and MnPII are (0.7 +/- 0.1) and (0.14 +/- 0.01) s-1, respectively, when the reaction is conducted in lactate buffer. Rate constants are considerably lower when the reactions are conducted in succinate buffer. Single turnover experiments confirmed that MnII serves as an obligatory substrate for MnPII and that both oxidized forms of the enzyme form productive complexes with MnII. Finally, these results suggest the alpha-hydroxy acids such as lactate facilitate the dissociation of MnIII from the enzyme.     

The kinetics of bivalent metal ion dissociation from myosin subfragments.

Bivalent metal ions have multiple roles in subunit association and ATPase regulation in scallop adductor-muscle myosin. To help elucidate these functions, the rates of Ca2+ and Mg2+ dissociation from the non-specific high-affinity sites on the regulatory light chains were measured and compared with those of rabbit skeletal-muscle myosin subfragments. Ca2+ dissociation had a rate constant of about 0.7 s-1 in both species, as measured by the time course of the pH change on EDTA addition. Mg2+ dissociation had a rate constant of 0.05 s-1, as monitored by its displacement with the paramagnetic Mn2+ ion. It is concluded that the exchange between Ca2+ and Mg2+ at the non-specific site, on excitation of both skeletal and adductor muscles, is too slow to contribute to the activation itself. The release of bivalent metal ions from the non-specific site is, however, the first step in release of the scallop regulatory light chain (Bennett & Bagshaw (1986) Biochem. J. 233, 179-186). In scallop myosin additional specific sites are present, which can bind Ca2+ rapidly, to effect activation of the ATPase. In the course of this work, Ca2+ dissociation from EGTA was studied as a model system. This gave rates of 1 s-1 and 0.3 s-1 at pH 7.0 and pH 8.0 respectively.     

The kinetics of the alkaline dissociation of myosin.

The rates of two processes in alkaline (pH 10.5–11.5) myosin solutions at 0 °C have been investigated: production of ionized tyrosine residues and production of light subunits. The progressive absorbance change is shown to result from a first-order irrevocable exposure to solvent and subsequent ionization of 40% of the tyrosine residues. Extrapolation to zero time gives the spectrophotometric ionization curve for native myosin; the pK of the abnormal tyrosines exceeds 12. Similarly, extrapolation to infinite time gives the curve for denatured myosin; the pK of the normal tyrosines (and of all tyrosines after denaturation) is 11.0–11.6. From the pH dependence of the rate, it is found that activation requires ionization of six residues and that their pK is much greater than 11.3. The rate of production of subunits was determined by fractionating the reaction mixture and determining the weight of light subunits produced. The process is also first order. Within experimental error, the rate constants for these two processes are equal. We conclude that they have the same rate-determining step. The data are consistent with either of two simple possible mechanisms. These are a rapid conformation change, followed by rate-determining subunit dissociation, followed by a rapid, irrevocable conformation change; or, a rapid conformation change, followed by a rate-determining, irrevocable conformation change, followed by rapid subunit dissociation.     

Transient spectroscopy of the reaction of cyanide with ferrous myoglobin. Effect of distal side residues

The reaction of cyanide metmyoglobin with dithionite conforms to a two-step sequential mechanism with formation of an unstable intermediate, identified as cyanide bound ferrous myoglobin. This reaction was investigated by stopped-flow time resolved spectroscopy using different myoglobins, i.e. those from horse heart, Aplysia limacina buccal muscle, and three recombinant derivatives of sperm whale skeletal muscle myoglobin (Mb) (the wild type and two mutants). The myoglobins from horse and sperm whale (wild type) have in the distal position (E7) a histidyl residue, which is missing in A. limacina Mb as well as the two sperm whale mutants (E7 His----Gly and E7 His----Val). All these proteins in the reduced form display an extremely low affinity for cyanide at pH less than 10. The differences in spectroscopy and kinetics of the ferrous cyanide complex of these myoglobins indicate a role of the distal pocket on the properties of the complex. The two mutants of sperm whale Mb are characterized by a rate constant for the decay of the unstable intermediate much faster than that of the wild type, at all pH values explored. Therefore, we envisage a specific role of the distal His (E7) in controlling the rate of cyanide dissociation and also find that this effect depends on the protonation of a single ionizable group, with pK = 7.2, attributed to the E7 imidazole ring. The results on A. limacina Mb, which displays the slowest rate of cyanide dissociation, suggests that a considerable stabilizing effect can be exerted by Arg E10 which, according to Bolognesi et al. (Bolognesi, M., Coda, A., Frigerio, F., Gatti, C., Ascenzi, P., and Brunori, M. (1990) J. Mol. Biol. 213, 621-625), interacts inside the pocket with fluoride bound to the ferric heme iron. A mechanism of control for the rate of dissociation of cyanide from ferrous myoglobin, involving protonation of the bound anion, is discussed.     

Dependence of co-operativity in the reaction of haemoglobin with oxygen on deuterium oxide concentration and temperature

Concentrated deionized solutions of haemoglobin in water were diluted with unbuffered pure ²H₂O and left to stand for 15 to 70 hours. Oxygen equilibrium curves were measured at different temperatures and concentrations of ²H₂O. In 98.5% ²H₂O Hill's constant retained its normal value at temperatures below 11 °C, but was reduced by 0.4 above 11 °C. This temperature effect was reversible. Lowering the ²H₂O concentrations raised the transition temperature between the states of low and high co-operativity of the reaction. The shape of the transition curve remained unchanged. Further experiments allowed a phase diagram to be constructed which shows the boundaries between the two states: one of low co-operativity at high concentration of ²H₂O and high temperature, and another of high co-operativity at low values of these variables. Reversibility of the isotope effect even at ²H₂O concentration of 98.5% excludes a purely steric interpretation. Possible dynamic and co-operative interactions between the protein molecule and its surrounding water molecules are discussed.     

The kinetics of ligand binding to plant hemoglobins. Structural implications

The rates of reaction of oxygen, carbon monoxide, and nitric oxide with 14 plant hemoglobins have been determined by relaxation and stopped-flow methods. The combination rates for oxygen lie between 0.12 and 0.26 x 10(9)/M.s, for carbon monoxide between 0.01 and 0.07 x 10(9)/M.s, and for nitric oxide between 0.12 and 0.25 x 10(9)/M.s. The dissociation velocities for oxygen range from 5 to 25/s, and for CO from 0.005 to 0.011 s. The oxygen dissociation constants range only from 36 to 78 nM. Nanosecond relaxation experiments show large differences between the proteins. Five have known primary structures which correlate closely with the nanosecond relaxations and less immediately with the millisecond reactions. The relevant amino acid substitutions are concentrated in the C-E interhelical region.     

Kinetic control of co-operativity in the oxygen binding of Panulirus interruptus hemocyanin.

The kinetics of the oxygen reaction of Panulirus interruptus hemocyanin have been studied at pH 9.6 under conditions where the protein exists in the undissociated, co-operative state and in the dissociated, non-co-operative state.Temperature-jump relaxation measurements of the undissociated protein at high oxygen saturation levels show one relaxation process which has been assigned to the high oxygen affinity (R) state, the on and off kinetic constants being 3.1 × 10⁷m^?1s^?1 and 60 s^?1, respectively. Stopped-flow measurements of the oxygen dissociation reaction show (1) an autocatalytic time-course of the reaction at pH 9.6 and (2) an increase in the overall oxygen dissociation rate constant, as the pH is decreased from 9.6 to 7.0.Temperature-jump relaxation measurements of the dissociated protein show one relaxation process characterized by a very high oxygen dissociation rate constant (1500 s^?1) and a combination constant which is of the same order of magnitude as reported for undissociated protein (k_on = 4.6 × 10⁷m^?1s${}^{\mathrm{?1}}$). The behaviour of dissociated protein can be considered as characteristic of the low oxygen affinity (T) state.The results presented in this paper, together with data available for other hemocyanins as well as hemoglobins, lead to the conclusion that respiratory proteins show a common feature in the kinetic control of co-operative oxygen binding: the stability of the oxygen-protein complex is largely determined by the value of the dissociation rate constant, the oxygen combination process very often appearing to be diffusion controlled.