©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cytochrome b of the Neutrophil Superoxide-generating System Contains Two Nonidentical Hemes
POTENTIOMETRIC STUDIES OF A MUTANT FORM OF gp91(*)

(Received for publication, March 27, 1995; and in revised form, April 6, 1995)

Andrew R. Cross (1)(§), Julie Rae (2), John T. Curnutte (2)

From the  (1)Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037 and the (2)Immunology Department, Genentech Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Analysis of potentiometric titrations of the cytochrome b from a X chronic granulomatous disease patient with an Arg Ser mutation in gp91 indicates that the mutant form of the cytochrome contains two nonidentical hemes with midpoint potentials of E = -220 and E = -300 mV. In the light of this information, reanalysis of redox titrations of wild-type cytochrome b implies that it probably also contains two separate heme centers with midpoint potentials of E = -225 and E = -265 mV. The effect of the Arg Ser substitution is to reduce the midpoint potential of one of the heme centers by approximately 35 mV and suggests possible interaction between Arg and a heme propionate side chain.


INTRODUCTION

Neutrophils contain a multicomponent, NADPH-dependent, O generating system that is used as a powerful antimicrobial weapon in the process of host defense (recently reviewed in (1) ). The redox centers of this NADPH oxidase, FAD and heme, are both contained within a unique low potential flavocytochrome, cytochrome b (also known as cytochrome b or cytochrome b) that also contains the NADPH binding domain(2, 3, 4) . Cytochrome b is composed of a heavily glycosylated large -subunit (gp91) and a small -subunit (p22), the products of two separately regulated genes. Each subunit is apparently unstable in the absence of its partner. The simplest model of cytochrome b is that of a gp91/p22 heterodimer containing 1 mol of heme and 1 mol of FAD. However, theoretical considerations and experimental evidence from purification(2, 5, 6, 7, 8) , proteolytic(9) , and reconstitution()(10, 11) studies suggest that each mole of cytochrome b probably contains 2 mol of heme and 1 mol of FAD. In addition, optical, EPR, CD, and resonance Raman spectra(7, 12, 13, 14, 15, 16) are consistent with the presence of multiple, bis-histidinyl, hexacoordinate, low spin hemes. Hitherto, the presence of only a single species of heme in cytochrome b has been inferred from oxidation-reduction potentiometric studies, where the cytochrome titrates as a single component with a midpoint potential of -245 mV (17) . Here we present evidence from the studies of the nonfunctional cytochrome from an X CGD()patient with an Arg Ser mutation in gp91that clearly demonstrates the presence of two nonidentical hemes with midpoint potentials of E = -220 mV and E = -300 mV. In light of this information, reanalysis of redox titrations of wild-type cytochrome b suggests the presence of two hemes with closely spaced midpoint potentials of E = -225 mV and E = -265 mV.


EXPERIMENTAL PROCEDURES

Materials

Anthraquinone 2,6-disulfonate, duroquinone, 2-hydroxy-1,4-naphthoquinone, and 2,3,5,6-tetramethylphenylenediamine were obtained from Aldrich. Anthraquinone and sodium dithionite were supplied by Fluka and Fisher, respectively; heptyl--D-thioglucopyranoside was from Calbiochem. All other reagents were purchased from Sigma. Pyocyanine was synthesized from phenazine methosulfate using the photochemical method described in (18) .

Isolation of Neutrophils and Preparation of Subcellular Fractions

Membranes and cytosolic fractions were prepared from the unstimulated neutrophils of both normal and CGD donors as described previously (19) and stored in aliquots at -80 °C.

Solubilization and Purification of Cytochrome b

Cytochrome b was partially purified as described previously(20) .

Measurement of Oxidation-Reduction Potentials

Potentiometric titrations were performed as described previously (17) using partially purified cytochrome b preparations derived from 9 10 neutrophils in a total volume of 2.7 ml of 100 mM KCl, 50 mM MOPS, pH 7.0. The following mediators were used at 12.5 µM: phenazine methosulfate, phenazine ethosulfate, anthraquinone, anthraquinone 2-sulfonate, anthraquinone 2,6-disulfonate, 2-hydroxy-1,4-naphthoquinone, 2,3,5,6-tetramethylphenylenediamine. Pyocyanine was added at 6 µM. Spectra were recorded between 580 and 520 nm at a series of electrode potentials, using a Uvikon 810 spectrophotometer. The degree of reduction of cytochrome b was estimated from the height of the absorbance band. An Orion 720A meter (Orion Research Inc., Boston MA) was used to measure the half-cell potential relative to a saturated calomel reference electrode. The potential was adjusted by the addition of <µl volumes of solutions of sodium dithionite (reductive titrations) and potassium ferricyanide (oxidative titrations). The accuracy of the apparatus was checked by titration of a 5 µM solution of phenosafranin (E = -252 mV).


RESULTS

We previously showed that the Arg Ser mutation in gp91 results in a cytochrome b with a nonfunctional heme with a slightly shifted Soret band that is unable to accept electrons from the reduced flavin center(21) . In contrast, the flavin domain of the cytochrome b is fully functional, as it is capable of accepting electrons from NADPH and reducing the artificial dye acceptor iodonitrotetrazolium violet at rates equivalent to that of the wild-type cytochrome. To determine if the loss of function was due to an alteration in heme redox potential, we performed a series of oxidation-reduction potential measurements using cytochrome b partially purified from neutrophil membranes from both normal and CGD patients. The results are shown in Fig. 1. The wild-type cytochrome titration could be fitted fairly well () to a Nernst equation curve for a single component with a E = -245 mV (Fig. 1A), the same midpoint potential as originally determined(17) . In contrast, titration of the Arg Ser cytochrome clearly did not fit the expected simple single component, 1-electron transfer process (Fig. 1B) (). By assuming two components are present, the data could be fitted to curves corresponding to two species with midpoint potentials of -220 and -300 mV, each contributing 50% to the absorbance at 559 nm (Fig. 2B) (). By inferring that two nonequivalent hemes are also present in the wild-type cytochrome b, the data in Fig. 1A can be fitted in a similar fashion to a 2-component curve with midpoint potentials of E = -225 and E = -265 mV (Fig. 2A) producing an excellent fit (). Titrations of the cytochrome b from a CGD patient with a Pro His mutation in gp91in which the flavin domain is nonfunctional (2, 21) were indistinguishable from that of the wild-type cytochrome, in accordance with a previous report (16) (data not shown).


Figure 1: Potentiometric titration of wild-type and Arg Ser cytochrome b; one component, 1-electron transfer. Oxidation-reduction potential measurements were performed as described under ``Experimental Procedures.'' Circles represent oxidative titrations (potassium ferricyanide); squares, reductive titrations (sodium dithionite). A, titration of wild-type cytochrome b; the solid curve is a theoretical line for a single species, 1-electron transfer process with a midpoint potential of -245 mV. B, titration of Arg Ser cytochrome b; the solid curve is the theoretical curve for a single species, 1-electron transfer with a midpoint of -260 mV.




Figure 2: Potentiometric titration of wild-type and Arg Ser cytochrome b; two components, 1-electron transfer. A, data as in Fig. 1A with the solid curve representing two species with midpoint potentials of -225 and -265 mV, each contributing 50% to the total absorbance change. B, data as in Fig. 1B with the solid curve representing two species with midpoint potentials of -220 and -300 mV, each contributing 50% to the total absorbance change. Circles represent oxidative titrations; squares, reductive titrations.




DISCUSSION

From the data presented above, it appears that both wild-type and mutant cytochromes contain two separate, nonidentical hemes. Both contain a heme center with a midpoint potential around -220 mV contributing approximately 50% to the total absorbance change; in the wild-type cytochrome, there is a second, closely spaced center with E = -265 mV. The similarity in redox potentials makes resolution of the two centers difficult in the wild-type cytochrome. In the Arg Ser cytochrome, the potential of the second lower potential center has been shifted downward by 35 mV from -265 mV to -300 mV and can be clearly resolved. At the present time, the locations of the hemes within the cytochrome are not known with any certainty. p22, which is reported to contain heme, contains only one invariant histidine, the latter lying within a sequence with homology to the heme-binding domains of a cytochrome oxidase subunit and the chromaffin granule cytochrome b. In view of the assignment of the axial ligands in cytochrome b as bis-histidinyl, a single p22 subunit cannot wholly contain a heme group. gp91 contains 17 histidines, none lying within sequences known to be homologous with other heme proteins. Thus, one heme group is probably shared between a p22 and gp91subunit (or two p22 subunits), and one heme is likely to be contained within gp91 itself.

It is thought that invariant arginine residues near the membrane surface of mitochondrial cytochromes b form hydrogen bonds with the negatively charged propionyl groups of the heme (22 and references therein). Hydropathy plots of gp91 predict Arg to be at the beginning of a membrane-spanning segment close to the membrane surface, and, therefore, this residue could perform a similar hydrogen bonding function in cytochrome b. The adjacent membrane-spanning segment (amino acids 100-120) contains 3 potential heme-liganding histidine residues. Substitution of the positively charged Arg to an uncharged serine residue would decrease the electron withdrawing nature of this group and thereby lower the redox potential of the heme. Site-directed mutagenesis studies of the analogous situation in iso-1-cytochrome c have demonstrated such decreases in redox potential when glutamine (-30 mV), asparagine (-34 mV), or alanine (-50 mV) were substituted for the arginine (Arg) that forms a hydrogen bond with one of the heme propionate side chains(23) .

In view of the fact that the redox potential of the heme in cytochrome b is altered by less than 40 mV by the Arg Ser substitution, it is perhaps surprising that the mutant form has no detectable O generating activity, particularly since we have shown that the flavin center is fully functional in this mutation(20, 21) . Further studies using site-directed mutagenesis and other naturally occurring mutant forms of cytochrome b will be invaluable in dissecting the factors affecting electron transfer functions in cytochrome b.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant AI24838, General Clinical Research Center Grant RR00833, and the Stein Endowment Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Molecular & Experimental Medicine CAL-1, The Scripps Research Institute, 10666 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 619-554-3654; Fax: 619-554-6988; scross{at}riscsm.scripps.edu

A. R. Cross, unpublished results.

The abbreviations used are: CGD, chronic granulomatous disease; MOPS, 4-morpholinepropanesulfonic acid; E, midpoint redox potential.


REFERENCES
  1. Thrasher, A. J., Keep, N. H., Wientjes, F., and Segal, A. W.(1994) Biochim. Biophys. Acta 1227, 1-24 [Medline] [Order article via Infotrieve]
  2. Segal, A. W., West, I., Wientjes, F., Nugent, J. H. A., Chavan, A. J., Haley, B., Garcia, R. C., Rosen, H., and Scrace, G.(1992)Biochem. J. 284, 781-788 [Medline] [Order article via Infotrieve]
  3. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., and Kwong, C. H.(1992) Science 256, 1459-1462 [Medline] [Order article via Infotrieve]
  4. Sumimoto, H., Sakamoto, N., Nozaki, M., Sakaki, Y., Takeshige, K., and Minakami, S. (1992)Biochem. Biophys. Res. Commun. 186, 1368-1375 [Medline] [Order article via Infotrieve]
  5. Harper, A. M., Dunne, M. J., and Segal, A. W.(1984)Biochem. J. 219, 519-527 [Medline] [Order article via Infotrieve]
  6. Parkos, C. A., Allen, R. A., Cochrane, C. G., and Jesaitis, A. J.(1987)J. Clin. Invest. 80, 732-742 [Medline] [Order article via Infotrieve]
  7. Bellavite, P., Cross, A. R., Serra, M. C., Davoli, A., Jones, O. T. G., and Rossi, F. (1983)Biochim. Biophys. Acta 746, 40-47 [Medline] [Order article via Infotrieve]
  8. Yoshida, L. S., Chiba, T., and Kakinuma, K.(1992)Biochim. Biophys. Acta 1135, 245-252 [Medline] [Order article via Infotrieve]
  9. Quinn, M. T., Mullen, M. L., and Jesaitis, A. J.(1992)J. Biol. Chem. 267, 7303-7309 [Abstract/Free Full Text]
  10. Koshkin, V., and Pick, E.(1994)FEBS Lett. 338, 285-289 [CrossRef][Medline] [Order article via Infotrieve]
  11. Doussière, J., Buzenet, G., and Vignais, P. V.(1995)Biochemistry 34, 1760-1770 [Medline] [Order article via Infotrieve]
  12. Iizuka, T., Kanegasaki, S., Makino, R., Tanaka, T., and Ishimura, Y.(1985)J. Biol. Chem. 260, 12049-12053 [Abstract/Free Full Text]
  13. Miki, T., Fujii, H., and Kakinuma, K.(1992)J. Biol. Chem. 267, 19673-19675 [Abstract/Free Full Text]
  14. Yamaguchi, T., Hayakawa, T., Kaneda, M., Kakinuma, K., and Yoshida, A.(1989) J. Biol. Chem. 264, 112-118 [Abstract/Free Full Text]
  15. Isogai, Y., Iizuka, T., Makino, R., Iyanagi, T., and Orii, Y.(1993)J. Biol. Chem. 268, 4025-4031 [Abstract/Free Full Text]
  16. Hurst, J. K., Loehr, T. M., Curnutte, J. T., and Rosen, H.(1991)J. Biol. Chem. 266, 1627-1634 [Abstract/Free Full Text]
  17. Cross, A. R., Jones, O. T. G., Harper, A. M., and Segal, A. W.(1981)Biochem. J. 194, 599-606 [Medline] [Order article via Infotrieve]
  18. Knight, M., Hartman, P. E., Hartman, Z., and Young, V. M.(1979)Anal. Biochem. 95, 19-23 [Medline] [Order article via Infotrieve]
  19. Curnutte, J. T., Kuver, R., and Babior, B. M.(1987)J. Biol. Chem. 262, 6450-6452 [Abstract/Free Full Text]
  20. Cross, A. R., and Curnutte, J. T.(1995)J. Biol. Chem. 270, 6543-6548 [Abstract/Free Full Text]
  21. Cross, A. R., Heyworth, P. G., Rae, J., and Curnutte, J. T.(1995)J. Biol. Chem. 270, 8194-8200 [Abstract/Free Full Text]
  22. Palmer, G., and Degli Esposti, M.(1994)Biochemistry 33, 176-185 [Medline] [Order article via Infotrieve]
  23. Cutler, R. L., Davies, A. M., Creighton, S., Warshel, A., Moore, G. R., Smith, M., and Mauk, A. G.(1989)Biochemistry 28, 3188-3197 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.