Ligand binding properties of cytochromes c'.

The cytochromes c' bind CO, alkylisocyanides and CN- with rate and equilibrium constants which are 10(2)- to 10(6)-fold smaller than other high-spin hemoproteins. The decreased affinity for exogenous ligands is largely associated with steric interactions at the heme coordination site. While CO and alkylisocyanides bind noncooperatively to the dimeric Rhodospirillum molischianum cytochrome c', CO, alkylisocyanides and CN- appear to bind cooperatively to the dimeric Chromatium vinosum cytochrome c' due to a ligand-linked dimer-monomer dissociation equilibrium. The differences between the cytochromes c' are thought to be due to differences in amino acid residues near the heme coordination site and subunit interface.     

Ligand-controlled dissociation of Chromatium vinosum cytochrome c'

Carbon monoxide binding to Chromatium vinosum ferrocytochrome c' has been studied by high-precision equilibrium methods. In contrast to the CO binding properties of Rhodospirillum molischianum cytochrome c' [Doyle, M. L., Weber, P. C., & Gill, S. J. (1985) Biochemistry 24, 1987-1991], CO binding to C. vinosum cytochrome c' is found to be unusual in the following ways. The binding curve is found to be cooperative with typical Hill coefficients equal to 1.25. The shape of the binding curve is asymmetrical. The heat of CO ligation is measured by two independent methods, both of which yield large endothermic values of approximately 10 kcal [mol of CO(aq)]-1. The overall affinity for CO increases as the concentration of cytochrome c' decreases. These observations suggest the CO binding properties of C. vinosum cytochrome c' are complicated by CO-linked association-dissociation processes. Further investigation by gel filtration chromatography shows that at micromolar concentrations the dimeric state is tightly associated in both the reduced and oxidized forms of the cytochrome but addition of saturating concentrations of CO causes the reduced ligated dimer to dissociate largely into monomers. A model is presented that quantitatively fits the data, involving a ligand-linked dimer-monomer dissociation reaction. In this model, CO binds to the dimer form noncooperatively with an intrinsic affinity constant equal to 5600 +/- 1200 M-1 at 25 degrees C. The unligated dimer form is tightly associated, but addition of CO causes dissociation of the dimer into the monomer with a monomer-dimer association constant equal to 450 +/- 200 M-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand-dependent Bohr effect of Chrionomus hemoglobins.

The O2 and CO Bohr effects of monomeric and dimeric hemoglobins of the insect Chironomus thummi thummi were determined as proton releases upon ligation. For the O2 Bohr effect of the monomeric hemoglobin III a maximum value of 0.20 H+/heme was obtained at pH 7.5. Upon ligation with CO, however, only 0.04 H+/heme were released at the same pH. In agreement with this finding isoelectric focusing experiments revealed different isoelectric points for O2-liganded and CO-liganded states of hemoglobin III. Analogous results were obtained in the cases of the monomeric hemoglobin IV and the dimeric hemoglobins of Chironomus thummi thummi; here O2 Bohr effects of 0.43 and 0.86 H+/heme were observed. For the corresponding CO Bohr effects values of 0.08 and 0.31 H+/heme were obtained respectively. On the basis of the available structural data the reduced CO Bohr effect in hemoglobin III is discussed as arising from a steric hindrance of the CO ligand by the side chain of isoleucine-E11, obstructing the movement of the heme-iron upon reaction with carbon monoxide. It should, however, be noted that ligands, according to their different electron donor and acceptor properties, may generally induce different conformational changes and thus different Bohr effects, in those hemoglobins in which distinct tertiary and/or quaternary constraints have not evolved. The general utilization of CO instead of O2 as allosteric effector is ruled out by the results reported here.     

Steric and hydrophobic effects in alkyl isocyanide binding to Rhodospirillum molischianum cytochrome c'

Equilibrium constants for the binding of a series of alkyl isocyanides to ferrous cytochrome c' from Rhodospirillum molischianum have been measured spectrophotometrically. The equilibrium constants range from 3.3 M-1 to 2.6 x 10(2) M-1 and follow the order methyl greater than ethyl less than n-propyl less than tert-butyl less than n-butyl less than amyl less than cyclohexyl less than n-hexyl. The decrease in equilibrium constant from methyl to ethyl isocyanide provides evidence for a steric interaction between the ligand and the protein. The increase in equilibrium constant from ethyl to n-hexyl isocyanide is accounted for by a favorable partitioning of the ligand into a hydrophobic heme coordination site. The effect of steric interactions on the differences in the binding constants has been further evaluated by comparing the alkyl isocyanide and CO binding constants for the ferrous cytochrome c' to those of a sterically unconstrained model heme complex in a detergent micelle. The results indicate that the heme coordination site of the ferrous cytochrome c' is severely sterically hindered, similar to that of the reported crystal structure of Rs. molischianum ferric cytochrome c'.     

Functional properties of partially oxidized trout hemoglobins.

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.     

Influence of carbon monoxide on hemoglobin-oxygen binding.

The oxygen dissociation curve and Bohr effect were measured in normal whole blood as a function of carboxyhemoglobin concentration [HbCO]. pH was changed by varying CO2 concentration (CO2 Bohr effect) or by addition of isotonic NaOH or HCl at constant PCO2 (fixed acid Bohr effect). As [HbCO] varied through the range of 2, 25, 50, and 75%, P50 was 26.3, 18.0, 11.6, and 6.5 mmHg, respectively. CO2 Bohr effect was highest at low oxygen saturations. This effect did not change as [HbCO] was increased. However, as [HbCO] was increased from 2 to 75%, the fixed acid Bohr factor increased in magnitude from -0.20 to -0.80 at very low oxygen saturations. The effect of molecular CO2 binding (carbamino) on oxygen affinity was eliminated at high [HbCO]. These results are consistent with the initial binding of O2 or CO to the alpha-chain of hemoglobin. The results also suggest that heme-heme interaction is different for oxygen than for carbon monoxide.     

Correlations between structural and spectroscopic properties of the high-spin heme protein cytochrome c'

The cytochromes c' are a class of heme proteins whose native spectroscopic properties have been suggested to represent a quantum mechanical admixture of intermediate-(S = 3/2) and high-(S = 5/2) spin states. Here features of the cytochrome c' heme environment, as revealed by X-ray crystallographic studies of the dimeric cytochrome c' from Rhodospirillum molischianum, are related to the observed spectroscopic properties. The environment of the heme group in cytochrome c' supports the existence of the admixed spin state at neutral pH and suggests that pH-dependent transition to a pure high-spin state at alkaline pH involves deprotonation of the histidine axial ligand to the heme iron.     

Involvement of the distal histidine in the low affinity exhibited by Hb Chico (Lys beta 66----Thr) and its isolated beta chains.

Hemoglobin (Hb) Chico (Lys beta 66----Thr at E10) has a diminished oxygen affinity (Shih, D. T.-b., Jones, R. T., Shih, M. F.-C., Jones, M. B., Koler, R. D., and Howard, J. (1987) Hemoglobin 11, 453-464). Our studies show that its P50 is about twice that of Hb A and that its cooperativity, anion, and Bohr effects between pH 7 and 8 are normal. The Bohr effect above pH 8 is somewhat reduced, indicating a small but previously undocumented involvement of the ionic bond formed by Lys beta 66 in the alkaline Bohr effect. Since the oxygen affinity of the alpha-hemes is likely to be normal, that of the beta-hemes in the tetramer is likely to be reduced by the equivalent of 1.2 kcal/mol beta-heme in binding energy. Remarkably, both initial and final stages of oxygen binding to Hb Chico are of lowered affinity relative to Hb A under all conditions examined. The isolated beta chains also show diminished oxygen affinity. In T-state Hb A, Lys(E10 beta) forms a salt bridge with one of the heme propionates, but comparison with other hemoglobin variants shows that rupture of this bridge cannot be the cause of the low oxygen affinity. X-ray analysis of the deoxy structure has now shown that Thr beta 66 either donates a hydrogen bond to or accepts one from His beta 63 via a bridging water molecule. This introduces additional steric hindrance to ligand binding to the T-state that results in slower rates of ligand binding. We measured the O2/CO partition coefficient and the kinetics of oxygen dissociation and carbon monoxide binding and found that lowered O2 and CO affinity is also exhibited by the R-state tetramers and the isolated beta chains of Hb Chico.     

Acid Bohr effects in myoglobin characterized by proton NMR hyperfine shifts and oxygen binding studies.

Proton NMR studies of sperm whale and horse deoxymyoglobin have revealed that both proteins exhibit a single, well defined, pH-induced structural change. The changes in hyperfine shifts are clearly observed not only at the heme peripheral substituents, but also at the proximal histidyl imidazole, which suggest that heme-apoprotein contacts are looser in the acidic than alkaline conformations. The hyperfine shift changes are modulated by a single titratable group with a pK of approx. 5.7 in both proteins. Oxygen binding studies of sperm whale myoglobin over a range of temperature and pH showed that, while the oxygen affinity was independent of pH at 25 degrees C, it increased below pH 7 at 0 degrees C and decreased below pH 7 at 37 degrees C. Hence, sperm whale myoglobin exhibits a small acid Bohr effect which most likely arises from the characterized structural changes in the deoxy proteins. While horse myoglobin failed to exhibit a resolvable acid Bohr effect between 0 and 37 degrees C, it did show a weak alkaline Bohr effect at 25 degrees C which disappeared at lower temperatures. Since the oxygen affinity changed smoothly over several pH units, this alkaline Bohr effect can not be associated with any well defined conformational change detected by NMR.     

Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase

Microbial cytochromes c' contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 A resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His-Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species. In contrast, CO binds 6-coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c' are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.     

Thermodynamics of carbon monoxide binding to monomeric cytochrome c'

The thermodynamic parameters for carbon binding to monomeric Rhodopseudomonas palustris cytochrome c' are determined. An enthalpy change for CO(aq) binding to the cytochrome is measured directly by titration calorimetry as -6.7 +/- 0.2 kcal/mol of heme, the CO binding equilibrium constant is measured at 35 degrees C as (1.96 +/- 0.05) X 10(5) M-1, and the binding equilibrium constant at 25 degrees C is calculated from the van't Hoff equation as (2.8 +/- 0.1) X 10(5) M-1. Comparison of the results to the known energetics of CO binding to dimeric cytochrome c', where the CO binding site is buried in the protein interior, indicates that the heme binding site on the monomer form is, in contrast, more exposed.     

Mechanism and biological role of nitric oxide binding to cytochrome c'

The binding of nitric oxide to ferric and ferrous Chromatium vinosum cytochrome c' was studied. The extinction coefficients for the ferric and ferrous nitric oxide complexes were measured. A binding model that included both a conformational change and dissociation of the dimer into subunits provided the best fit for the ferric cytochrome c' data. The NO (nitric oxide) binding affinity of the WT ferric form was found to be comparable to the affinities displayed by the ferric myoglobins and hemoglobins. Using an improved fitting model, positive cooperativity was found for the binding of NO to the WT ferric and ferrous forms, while anticooperativity was the case for the Y16F mutant. Structural explanations accounting for the binding are proposed. The NO affinity of ferrous cytochrome c' was found to be much lower than the affinities of myoglobins, hemoglobins, and pentacoordinate heme models. Structural factors accounting for the difference in affinities were analyzed. The NO affinity of ferrous cytochrome c' was found to be in the range typical of receptors and carriers. In addition, cytochrome c' was found to react with cytosolic light-irradiated membranes in the presence of succinate and carbon monoxide. With these results, a biochemical model of cytochrome c' functioning as a nitric oxide carrier was proposed.     

Stoichiometry determination for carbon monoxide binding to Rhodospirillum molischianum cytochrome c'

The stoichiometry of CO ligation to the dimer heme protein Rhodospirillum molischianum cytochrome c' is determined. We have recently measured the enthalpy change of CO ligation to this molecule by the van't Hoff method and found the value of -10.7 +/- 1.2 kcal/mol CO (aqueous) (Doyle, M. L., Weber, P. C., and Gill, S. J. (1985) Biochemistry 24, 1987-1991). In the present paper the enthalpy change of CO ligation, measured directly by titration calorimetry, is found to be -9.5 +/- 0.2 kcal/mol heme. Since the van't Hoff method gives the heat value in units/mole of CO and the calorimetric method gives the heat value in units/mole of heme, the stoichiometry of the reaction is given by the ratio of the two values and found to be 0.9 +/- 0.1, or within experimental error, one CO molecule bound per heme.     

Ligand-induced monomerization of <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>′ studied using native mass spectrometry and fluorescence resonance energy transfer

Cytochrome c' from Allochromatium vinosum is an attractive model protein to study ligand-induced conformational changes. This homodimeric protein dissociates into monomers upon binding of NO, CO or CN(-) to the iron of its covalently attached heme group. While ligand binding to the heme has been well characterized using a variety of spectroscopic techniques, direct monitoring of the subsequent monomerization has not been reported previously. Here we have explored two biophysical techniques to simultaneously monitor ligand binding and monomerization. Native mass spectrometry allowed the detection of the dimeric and monomeric forms of cytochrome c' and even showed the presence of a CO-bound monomer. The kinetics of the ligand-induced monomerization were found to be significantly enhanced in the gas phase compared with the kinetics in solution, however. Ligand binding to the heme and the dissociation of the dimer in solution were also studied using energy transfer from a fluorescent probe to both heme groups of the protein. Comparison of ligand binding kinetics as observed with UV-vis spectroscopy with changes in fluorescence suggested that binding of one CO molecule per dimer could be sufficient for monomerization.     

Reactivity of ferrous myoglobin at low pH.

The rates of reaction of myoglobin with carbon monoxide at low pH are reported. The pH versus rate profile of these kinetics resembles that found for heme model compounds, revealing an increase in combination rate at low pH. These facts suggest that CO binding by myoglobin changes from a mechanism of "direct ligant association" at pH 5 to a mechanism, similar to that proposed for heme model compounds, which assumes a tetracoordinated intermediate as a result of the protonation of the proximal imidazole.     

Co-binding studies on Hb M Iwate. Allostery of a T state haemoglobin.

The mutant haemoglobin Hb M Iwate alpha 2Mmet87His leads to Tyr beta 2, is characterized by a stable T structure and a low ligand affinity. Sigmoidal CO-binding isotherms of symmetrical shape with Hill coefficients of n = 1.4 at pH 6 to n = 1.9 at pH 10 and the differences in the mean affinity (PCO(1/2)) and the affinity of the first ligand-binding beta subunit (1/L1 greater than Pco(1/2)) are the evidence for the cooperativity. The comparison of the Bohr effects of the two valency hybrid states (alpha 2Mmet beta met beta deoxy alpha 2Mmet beta 2deoxy) in the absence of and in the presence of polyphosphates leads to an indirect proof of pH-dependent subunit-subunit interaction. Inositol hexaphosphate-binding suppresses cooperativity in the pH range 5.5-8 (n = 1). Above pH 8 hte cooperativity increases to a final value of n = 1.9 at pH greater than 10, which is identical to that of stripped Hb M Iwate. The CO binding to the first binding site exhibits a Bohr effect. Polyphosphate anions have no influence on the CO binding of the first binding site. The heterotropic effects are discussed as intrachain effects (Bohr effect of the first binding site) and interchain effects (Bohr effect of Pco(1/2); influence of polyphosphates).     

Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin

Rate constants have been measured for the reactions of a series of high-spin cytochromes c' and their low-spin homologues (cytochromes c-554 and c-556) with the semiquinones of free flavins and flavodoxin. These cytochromes are approximately 3 times more reactive with lumiflavin and riboflavin semiquinones than are the c-type cytochromes that are homologous to mitochondrial cytochrome c. We attribute this to the greater solvent exposure of the heme in the c'-type cytochromes. In marked contrast, the cytochromes c' are 3 orders of magnitude less reactive with flavodoxin semiquinone than are the c-type cytochromes. We interpret this result to be a consequence of the location of the exposed heme in cytochrome c' at the bottom of a deep groove in the surface of the protein, which is approximately 10-15 A deep and equally as wide. While free flavins are small enough to enter the groove, the flavin mononucleotide (FMN) prosthetic group of flavodoxin is apparently prevented by steric constraints from approaching the heme more closely than approximately 10 A without dynamic structural rearrangements. Most cytochromes c' are dimeric, but a few are monomeric. The three-dimensional structure of the Rhodospirillum molischianum cytochrome c' dimer suggests that the heme should be more exposed in the monomer than in the dimer, but no relationship is observed between intrinsic reactivity toward free flavin semiquinones and the aggregation state of the protein. Likewise, there is no evidence that the spin state or ligand field of the iron has any effect on intrinsic reactivity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton activities for three states of cytochrome P-450cam.

Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the $\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.     

Functional properties of hemoglobins from deep-sea fish: correlations with depth distribution and presence of a swimbladder

The ligand binding properties of the hemoglobins of several deep-sea, bottom-living fish have been examined. These include five species of rattails (Macrouridae) and Antimora rostrata, all of which possess swimbladders, and two unrelated species without swimbladders, Bathysaurus mollis and Alepocephalus sp. All of the hemolysates of these fish exhibited the Root effect with a minimum ligand affinity at about pH 6 in the presence of organic phosphate. Under these conditions the hemolysates from fish which possess swimbladders exhibit two roughly equal populations of heme groups with markedly different ligand affinities. For the deeper-dwelling species the affinities for carbon monoxide differ by some 500-fold, the low-affinity population having a p50(CO) of 100 mmHg at 15 degrees C. This very low affinity is associated with a second-order rate constant for CO combination of the order of 10(3) M-1 X s-1. Those species without swimbladders have hemoglobins which do not have such heterogeneous binding sites, suggesting a relationship between these very-low-affinity heme groups and the pumping of oxygen into a swimbladder at high hydrostatic pressures.     

Functional properties of the hemoglobin from the South American snake Mastigodryas bifossatus

The hemolysate of Mastigodryas bifossatus shows two major hemoglobins with very close isoelectric points, and four different globin chains. The stripped hemolysate exhibits a low alkaline Bohr effect (Δlog P₅₀/ΔpH = −0.30 between pH7 and 8) and a decrease of the co-operativity from 2.3 to unity when the pH increases from 6.15 to 8.5. In the presence of ATP, large changes in the oxygen affinity and co-operativity are observed. The Bohr effect rises to −0.46 and the n₅₀ values stay at around 3 in the pH range 6–9. An increase in temperature induces a large decrease in the oxygen affinity for the stripped hemolysate. In the pH range between 7.5 and 8.5, the values of AH in kcal/M are around 10 fold larger for the stripped protein than for the protein in the presence of ATP. Measurements of rapid kinetics of oxygen dissociation and carbon monoxide binding reflect the ATP sensitivity observed in equilibrium experiments.