Long-range reactive dynamics in myoglobin.

We report the complete vibrational spectrum of the probe nucleus 57Fe at the oxygen-binding site of the protein myoglobin. The Fe-pyrrole nitrogen stretching modes of the heme group, identified here, probe asymmetric interactions with the protein environment. Collective oscillations of the polypeptide, rather than localized heme vibrations, dominate the low frequency region. We conclude that the heme "doming" mode is significantly delocalized, so that distant sites respond to oxygen binding on vibrational time scales. This has ramifications for understanding long-range interactions in biomolecules, such as those that mediate cooperativity in allosteric proteins.

Isoelectrophoretic characterization of Pseudomonas cytochrome oxidase/nitrite reductase and its heme d1-containing domain.

The cytochrome oxidase/nitrite reductase of Pseudomonas aeruginosa has been purified to homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When this "homogeneous" protein is subjected to electrophoretic titration curve analysis in ampholines or to isoelectric focusing in immobilized pH gradient gels it is resolved into several bands, each of which possesses the olive-green color of the holoenzyme. Although the patterns of resolution replicate for a given enzyme preparation differences occur among different preparations. Furthermore, storage for several months at -20 degrees C leads to an increase in the number of isoelectrophoretic forms. All preparations, however, have two primary bands, one with a pI of 6.97 and the other of 7.02. Both these bands possess significant cytochrome oxidase activity after elution from the gels. When each of the primary bands is eluted and again subjected to isoelectric focusing under the same conditions as before, each band interconverts into two bands with pIs of 6.97 and 7.02. The addition of the ligand cyanide to the holoenzyme produces a shift in the pI of the two bands to pIs 7.04 and 7.12 while the addition of nitrite shifts some of the band at pI 6.97 into that at pI 7.02. The heme d1-containing dipeptide of the enzyme, produced by treatment with subtilisin, also exhibits considerable heterogeneity upon electrophoretic titration curve analysis and by isoelectric focusing in immobiline gels. Possible explanations for the observed isoelectrophoretic behavior in terms of protein conformation and heme chemistry are discussed.

Perturbation of Pseudomonas cytochrome oxidase by guanidine hydrochloride to detect differential stabilization of the heme d1 and heme c moieties.

The optical properties of Pseudomonas cytochrome oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.2) were monitored as a function of guanidine hydrochloride (Gdn X HCl) concentration to probe for differential stabilization of its prosthetic groups, heme d1 and heme c. The protein fluorescence intensity increased with the Gdn X HCl concentration, revealing two transitions, a sharp one between 1.3 and 1.5 M Gdn X HCl, and a second less well defined extending from 2.5 to 4.5 M. Only the transition at the lower Gdn X HCl concentrations was present in titrations followed using the emission maxima. The spectral maximum for native Pseudomonas cytochrome oxidase was at approx. 335 nm and shifted to approx. 350 nm above 2 M Gdn X HCl. The heme d1 absorbance at 638 nm decreased with increasing [Gdn X HCl], giving a transition at 1.3-1.5 M, and no transition up to 4 M Gdn X HCl when the heme c was monitored at 525 nm. Along with the decrease at 638 nm, an absorption band appeared at 681 nm, suggesting heme d1 release into solution. Fluorescence titration of heme d1-depleted enzyme, prepared by gel filtration, showed a single transition similar to the transition occurring in the intact enzyme at high Gdn X HCl concentrations. Circular dichroism spectra revealed clearly distinguishable transitions for the heme d1 and heme c near 1.5 and 3.0 M Gdn X HCl, respectively. These results suggest that the two hemes are in regions of the protein with different stabilities which may represent distinct structural domains.

Mta, the maternally transmitted antigen, is determined jointly by the chromosomal Hmt and the extrachromosomal Mtf genes.

Mus spretus from four stocks, originating in Spain, Portugal, and Morocco, were tested for the maternally transmitted antigen, Mta. All expressed a variant form not found in other species of mice. Analysis of appropriate crosses with inbred mice showed that the spretus form of Mta is determined by a new allele, c, of the Hmt gene. The Hmtc allele has been isolated in coupling with four different H-2 haplotypes. It is possible to raise CTL specific for the spretus form of Mta. The maternally transmitted factor, Mtf alpha s, of spretus mice determines, in conjunction with the Hmta allele of C57BL/6, an Mta that is indistinguishable from the common form found in C57BL/6 and most other inbred mice. Our experiments show that the specificity of the cell surface antigen Mta is governed jointly by the cytoplasmic gene Mtf and the chromosomal gene Hmt. We propose that Hmt encodes a class I histocompatibility antigen that acts as a restricting element for the Mtf gene product, thus meeting the requirements of T killer cell recognition.

Variations in the oxidation-reduction behavior of liganded species of Pseudomonas cytochrome oxidase.

In an effort to determine the steady-state redox properties of Pseudomonas aeruginosa cytochrome cd1, changes in absorption spectra after the addition of excess reductant (ascorbate, ferrous ethylenediaminetetraacetic acid) were monitored for degassed unliganded enzyme and samples in the presence of CO and CN- at pH 6.0, 8.0, or 10.0. Plots of [c2+]/[c3+] vs. [d2+]/[d3+] indicate that a "pseudoequilibrium" was reached for all samples at pH 8.0. Calculated values of delta Ed-c, the difference in reduction potential between the heme c and heme d moieties, at pH 8.0 were -25 +/- 5 (unliganded), -10 +/- 5 (enzyme-CO), and -25 +/- 5 mV (enzyme-CN). Relative rates of heme c and heme d reduction were found to be dependent upon type of ligand, reductant, and pH. Evidence for a cooperative heme c-heme d interaction is discussed.

Activation of Pseudomonas cytochrome oxidase by limited proteolysis with subtilisin.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c2: oxygen oxidoreductase; E.C.1.9.3.2) can be digested with subtilisin under controlled conditions that convert the original parent polypeptide chain (Mr on SDS gels approximately equal to 60,000) to a slightly smaller species (Mr on SDS gels approximately equal to 58,000). Under the conditions used (0.33% subtilisin, w/w, pH 7.4), the product formed from the oxidase was relatively stable to further digestion. Cytochrome oxidase activity was assayed at intervals during proteolysis by following the rate of oxidation of Pseudomonas ferrocytochrome c-551 by the enzyme in the presence of oxygen. The activity increased to a plateau that was more than two times the value for an untreated control. These observations suggest that clipping a small peptide from Pseudomonas cytochrome oxidase either facilitates the rate-limiting electron transfer between the intraprotein heme c and heme d1, enhances the interaction of the enzyme with ferrocytochrome c-551, or both.

The effects of copper depletion on structural aspects of cytochrome c oxidase.

The removal of copper from beef heart cytochrome c oxidase by either dialysis against potassium cyanide or by treatment with bathocuproine sulfonate produced changes in the enzyme which are indicative of a spin state transition. In the Soret region of the CD spectrum copper depletion of the enzyme caused a significant decrease in amplitude in combination with a red shift of the peak maximum for oxidized samples, while reduced copper-depleted samples exhibited decreased amplitude and a blue shift of the peak maximum. In the magnetic CD spectra of oxidized copper-depleted samples the peak at 420 nm was shifted to lower wave-length along with a significant increase in amplitude. In reduced samples the peak at 446 nm exhibited a slight red shift concomitant with a substantial decrease in amplitude. The conformational changes indicated by the CD and magnetic CD spectra when copper is removed from the enzyme were supported by the EPR spectra of the NO complex of the reduced copper-depleted enzyme. The removal of copper from cytochrome c oxidase caused the NO complex to exhibit a 3-line splitting pattern of gz in the EPR spectrum instead of the 9 lines seen in the NO complex of the native enzyme. When [15N]NO was used, a 2-line pattern was seen at gz when copper was removed from the enzyme. The changes in the CD and magnetic CD spectra and in the EPR spectra of the NO derivatives of cytochrome c oxidase can be explained by the rearrangement of the axial ligands to iron in cytochrome a3 as a result of copper depletion. These results emphasize the close structural interdependence of the metallic components of this enzyme.

Controlled proteolysis by subtilisin as a probe for cyanide-induced conformational changes in Pseudomonas cytochrome oxidase.

Oxidized Pseudomonas cytochrome oxidase (ferrocytochrome c-551:oxidoreductase; EC 1.9.3.2), digested with subtilisin in the absence and presence of KCN, produces discrete, high molecular weight fragments. The presence of KCN alters the rate of this fragmentation but does not change the nature of the fragments. When digested in the absence of KCN, the oxidase gives a major product (A) which is enzymatically active and has an apparent Mr = 58,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the presence of KCN, the major product (B) has Mr = 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis but with gel permeation high performance liquid chromatography it has an apparent Mr = 92,000. This implies that product B is dimeric, as is the parent enzyme which has Mr = 110,000 by high performance liquid chromatography. Absorption spectra of product B, isolated by gel filtration, show that it contains only the heme d1 moiety. The digestion time course indicates that the rate at which several minor products are formed is also dependent on the absence or presence of KCN. These observations suggest that the binding of KCN to the heme centers induces a conformational change in the enzyme so that the heme c-containing portion of the protein, which is at one end of the intact enzyme, can be removed without disrupting the integrity of the heme d1-containing portion.

Some redox properties of myohemerythrin from retractor muscle of Themiste zostericola.

Distinct semimetmyohemerythrin species are produced by one-electron oxidation of deoxymyohemerythrin and one-electron reduction of metmyohemerythrin. The former, (semimetmyo)o, changes (greater than or equal to 90%) to the latter, (semimetmyo)R, with k = 1.0 x 10(-2) s-1, delta H = 15.1 kcal mol-1 and delta S = -17 eu. Oxidation of (semimetmyo)o by Fe(CN)6(3)- rapidly produces an unstable metmyohemerythrin form which converts to the final metmyohemerythrin with k = 4.6 x 10(-3) s-1, delta H = 16.8 kcal mol-1, and delta S = -13 eu. The two met forms react at the same rate with N3-, but the unstable form reacts very rapidly with S2O4(2-) in contrast to stable metmyohemerythrin. (Semimetmyo)R or a mixture of metmyohemerythrin and deoxymyohemerythrin equilibrate very slowly to a mixture containing all three species. The rate constants for disproportionation and comproportionation are 0.89 M-1 s-1 and 9.4 M-1 s-1, respectively. EPR spectra near liquid He temperatures and optical absorption spectra have been used to characterize and measure the rates at 25 degrees C, pH 8.2, and I = 0.15 M. The comparative behavior of octameric and monomeric protein is discussed.

EPR spectroscopy of semi-methemerythrin.

EPR spectra of semi-met forms of octameric hemerythrin from Themiste zostericola, prepared by one electron reduction of methemerythrin or by one electron oxidation of deoxyhemerythrin, have been visualized at liquid helium temperatures. The spectrum of that prepared by one electron reduction has principal g-values of 1.96 +/- 0.01, 1.88 +/- 0.01, and 1.67 +/- 0.02 while that obtained by one electron oxidation has g = 1.95 +/- 0.01, 1.72 +/- 0.01, and 1.68 +/- 0.02. The amplitude of either spectrum decreases with time on incubation at room temperature according to a first order rate with t 1/2 = 5-8 min, apparently because of an intramolecular disproportionation. Similar EPR spectra have been obtained with semi-metmyohemerythrin of T. zostericola and with the octameric semi-met form of Phascolopsis gouldii. However, these forms disproportionate to a much lesser degree. The azide adduct of the octameric semi-met form of T. zostericola has g-values of 1.94 +/- 0.01, 1.85 +/- 0.01, and 1.57 +/- 0.02. Its EPR spectrum differs somewhat from those of the azide adducts of the octamer of P. gouldii and the monomer of T. zostericola although all are resistant to disproportionation. Methemerythrin and deoxyhemerythrin have no EPR spectra even at liquid helium temperature.

EPR study of heme x NO complexes of ascorbic acid-reduced Pseudomonas cytochrome oxidase and corresponding model complexes.

The EPR spectra of the NO complexes of frozen solutions of ascorbic acid-reduced cytochrome oxidase (nitrite reductase) purified from Pseudomonas aeruginosa, of its heme d1-depleted form, and of heme d1 in solutions containing various nitrogenous bases are quite similar to each other as well as to several heme (iron protoporphyrin IX)-containing proteins. The NO complexes of heme d1 (an iron-chlorin) in the presence of nitrogenous bases belong to spectral type C according to Kon's classification and, thus, the energy levels of the iron are closely related to thorse of heme complexes recorded under similar conditions. Comparison of these spectra with those of complexes of known structure suggests that both heme c and heme d1 are linked with Pseudomonas cytochrome oxidase by means of a nitrogenous ligand. The EPR spectrum of the NO complex of the native enzyme exhibits a lack of resolution of the high field (gy) resonance which can be characterized in terms of a spectral contribution from both the heme c and heme d1 moieties. The similarity between the EPR spectra of the NO complexes of horse heart cytochrome c and the heme d1-depleted Pseudomonas cytochrome oxidase before and after interaction with urea suggests structural similarities involving the heme irons. The changes caused by urea are likely to be a breaking or distortion of the bond between the iron and the protein-donated nitrogenous ligand and are similar to alterations seen with NO complexes of hemoglobin under a variety of conditions.

Small-angle X-ray scattering studies of oxidized and reduced cytochrome oxidase from Pseudomonas aeruginosa.

Small-angle X-rays scattering experiments were performed with oxidized and reduced cytochrome oxidase purified from Pseudomonas aeruginosa. The radii of gyration were calculated to be 40.5 A for the oxidized form and 37.0 A for the reduced. The longest dimension of the oxidized enzyme was 120 A while for the reduced it was 100 A. The volume of the oxidized protein was observed to be slightly greater than that of the reduced. These data indicate that there is a contraction of the structure of the enzyme during reduction of its constituent heme groups.

Detection of conformational changes in complex III of the respiratory chain by a maleimido spin label.

Changes in the conformation of Complex III (CoQH2-cytochrome c reductase) of the mitochondrial respiratory chain were detected upon oxidoreduction using the nitroxide spin label, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl. EPR spectra of the spin label show a transition from a greater to a lesser degree of immobilization when the labeled enzyme, reduced either with ascorbate or sodium dithionite, is oxidized with potassium ferricyanide or ferricytochrome c. These observations are interpreted to indicate that Complex III is more compact in the reduced state at least in the locality of the spin label. An apparent increase in the concentration of total spins during oxidation of the complex suggests change in the interaction between the spin label and other paramagnetic centers and not an oxidation of spin label, itself, since reduced free spin label could not be reoxidized. Addition of antimycin A had no effect on the EPR spectrum of the spin-labeled enzyme, indicating that this inhibitor does not initiate a conformational change in the region of the spin label. Experiments in which N-ethyl-[2-3H] maleimide was bound to Complex III show that binding occurs primarily to a subunit with a molecular weight of 45,000. Although no qualitative differences were observed, it was found that less radioactivity appears in samples reduced with dithionite than in those reduced with ascorbate. This difference appears to be caused by decomposition products of dithionite.

Reconstitution of the apoenzyme of cytochrome oxidase from Pseudomonas aeruginosa with heme d1 and other heme groups.

Cytochrome oxidase (EC 1.9.3.2) from Pseudomonas aeruginosa contains heme d1 and heme c in an equimolar ratio. The heme d1 can be removed from the enzyme with acidified acetone leaving an apoenzyme that contains heme c but has no oxidase activity. Reconstitution of the apoenzyme in neutral 6 M urea with heme d1 yields a reconstituted product which, after removal of the urea, has 90 to 100% of the oxidase activity of the native enzyme, a 1:1 molar ratio of the heme groups, and is indistinguishable from the native on the basis of its absorption spectral properties and its EPR spectrum. The apoenzyme can also be reconstituted with heme a, deuteroheme, hematoheme, mesoheme, and protoheme but only the heme a yields a product with any oxidase activity. The properties of these reconstituted products are compared.