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. 1989 Jan 1;257(1):87–94. doi: 10.1042/bj2570087

Studies on electron transfer from methanol dehydrogenase to cytochrome cL, both purified from Hyphomicrobium X.

M Dijkstra 1, J Frank Jr 1, J A Duine 1
PMCID: PMC1135541  PMID: 2537627

Abstract

Ferricytochrome cL isolated from Hyphomicrobium X is an electron acceptor in assays for homologous methanol dehydrogenase (MDH), albeit a poor one compared with artificial dyes. The intermediates of MDH seen during the reaction are identical with those observed with Wurster's Blue as electron acceptor, indicating that the reaction cycles are similar. The assay showed a pH optimum of approx. 7.0 and scarcely any stimulation by NH4Cl, this being in contrast with assays with artificial dyes, where strong activation by NH4Cl and much higher pH optima have been reported. From the results obtained with stopped-flow as well as steady-state kinetics, combined with the isotope effects found for C2H3OH, it appeared that the dissimilarities between the electron acceptors can be explained from different rate-limiting steps in the reaction cycles. Ferricytochrome cL is an excellent oxidant of the reduced MDH forms at pH 7.0, but the substrate oxidation step is very slow and the activation by NH4Cl is very poor at this pH. At pH 9.0 the reverse situation exists: ferricytochrome cL is a poor oxidant of the reduced forms of MDH at this pH. No C2H3OH isotope effect was observed under these conditions, indicating that substrate oxidation is not rate-limiting, so that activation by NH4Cl cannot be found. Since just the opposite holds for assays with artificial dyes, the poor electron-acceptor capability and the different pH optimum of ferricytochrome cL as well as the insignificant activating effect of NH4Cl (all compared with artificial assays) can be explained. Although different views have been reported on the rate-limiting steps in the systems from Methylophilus methylotrophus and Methylobacterium sp. strain AM1, these are most probably incorrect, as rate-limiting electron transfer between ferrocytochrome cL and horse heart ferricytochrome c can occur. Therefore the conclusions derived for the Hyphomicrobium X system might also apply to the systems from other methylotrophic bacteria. Comparison of the assays performed in vitro (at pH 7.0) having ferricytochrome cL and Wurster's Blue as electron acceptor with methanol oxidation by whole cells shows that the former has similarity whereas the latter has not, this being although ferricytochrome cL is a poor electron acceptor in the assay performed in vitro. The reason for this is the absence of a (natural) activator able to activate the (rate-limiting) substrate oxidation step at physiological pH values.

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Selected References

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  1. Alefounder P. R., Ferguson S. J. A periplasmic location for methanol dehydrogenase from Paracoccus denitrificans: implications for proton pumping by cytochrome aa3. Biochem Biophys Res Commun. 1981 Feb 12;98(3):778–784. doi: 10.1016/0006-291x(81)91179-7. [DOI] [PubMed] [Google Scholar]
  2. Anthony C. Bacterial oxidation of methane and methanol. Adv Microb Physiol. 1986;27:113–210. doi: 10.1016/s0065-2911(08)60305-7. [DOI] [PubMed] [Google Scholar]
  3. Anthony C. The microbial metabolism of C1 compounds. The cytochromes of Pseudomaonas AM1. Biochem J. 1975 Feb;146(2):289–298. doi: 10.1042/bj1460289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carver M. A., Humphrey K. M., Patchett R. A., Jones C. W. The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus. Eur J Biochem. 1984 Feb 1;138(3):611–615. doi: 10.1111/j.1432-1033.1984.tb07958.x. [DOI] [PubMed] [Google Scholar]
  5. Cheddar G., Meyer T. E., Cusanovich M. A., Stout C. D., Tollin G. Electron-transfer reactions between flavodoxin semiquinone and c-type cytochromes: comparisons between various flavodoxins. Biochemistry. 1986 Oct 21;25(21):6502–6507. doi: 10.1021/bi00369a024. [DOI] [PubMed] [Google Scholar]
  6. Dijkstra M., Frank J., Jr, van Wielink J. E., Duine J. A. The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL. Biochem J. 1988 Apr 15;251(2):467–474. doi: 10.1042/bj2510467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Duine J. A., Frank J., Jr Studies on methanol dehydrogenase from Hyphomicrobium X. Isolation of an oxidized form of the enzyme. Biochem J. 1980 Apr 1;187(1):213–219. doi: 10.1042/bj1870213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Duine J. A., Frank J., Westerling J. Purification and properties of methanol dehydrogenase from Hyphomicrobium x. Biochim Biophys Acta. 1978 Jun 9;524(2):277–287. doi: 10.1016/0005-2744(78)90164-x. [DOI] [PubMed] [Google Scholar]
  9. Eisenthal R., Cornish-Bowden A. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem J. 1974 Jun;139(3):715–720. doi: 10.1042/bj1390715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frank J., Jr, Dijkstra M., Duine J. A., Balny C. Kinetic and spectral studies on the redox forms of methanol dehydrogenase from Hyphomicrobium X. Eur J Biochem. 1988 Jun 1;174(2):331–338. doi: 10.1111/j.1432-1033.1988.tb14102.x. [DOI] [PubMed] [Google Scholar]
  11. Ghosh R., Quayle J. R. Purification and properties of the methanol dehydrogenase from Methylophilus methylotrophus. Biochem J. 1981 Oct 1;199(1):245–250. doi: 10.1042/bj1990245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldkorn T., Schejter A. Kinetic and spectroscopic evidence for different forms of ferric cytochrome c at very low ionic strength and neutral pH. FEBS Lett. 1977 Mar 15;75(1):44–46. doi: 10.1016/0014-5793(77)80049-5. [DOI] [PubMed] [Google Scholar]
  13. Goldkorn T., Schejter A. The reaction of ferric cytochrome c with dithionite at very low ionic strength. FEBS Lett. 1977 Oct 15;82(2):293–296. doi: 10.1016/0014-5793(77)80605-4. [DOI] [PubMed] [Google Scholar]
  14. Kasprzak A. A., Steenkamp D. J. Localization of the major dehydrogenases in two methylotrophs by radiochemical labeling. J Bacteriol. 1983 Oct;156(1):348–353. doi: 10.1128/jb.156.1.348-353.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. König B. W., Wilms J., Van Gelder B. F. The reaction between cytochrome c1 and cytochrome c. Biochim Biophys Acta. 1981 Jun 12;636(1):9–16. doi: 10.1016/0005-2728(81)90069-4. [DOI] [PubMed] [Google Scholar]
  16. Massey V., Hemmerich P. Photoreduction of flavoproteins and other biological compounds catalyzed by deazaflavins. Biochemistry. 1978 Jan 10;17(1):9–16. doi: 10.1021/bi00594a002. [DOI] [PubMed] [Google Scholar]
  17. O'Keeffe D. T., Anthony C. The interaction between methanol dehydrogenase and the autoreducible cytochromes c of the facultative methylotroph Pseudomonas AM1. Biochem J. 1980 Aug 15;190(2):481–484. doi: 10.1042/bj1900481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ohta S., Tobari J. Two cytochromes c of Methylomonas J. J Biochem. 1981 Jul;90(1):215–224. doi: 10.1093/oxfordjournals.jbchem.a133452. [DOI] [PubMed] [Google Scholar]
  19. Patchett R. A., Jones C. W. The apparent oxidation of NADH by whole cells of the methylotrophic bacterium Methylophilus methylotrophus. A cautionary tale. Antonie Van Leeuwenhoek. 1986;52(5):387–392. doi: 10.1007/BF00393466. [DOI] [PubMed] [Google Scholar]
  20. Simondsen R. P., Weber P. C., Salemme F. R., Tollin G. Transient kinetics of electron transfer reactions of flavodoxin: ionic strength dependence of semiquinone oxidation by cytochrome c, ferricyanide, and ferric ethylenediaminetetraacetic acid and computer modeling of reaction complexes. Biochemistry. 1982 Dec 7;21(25):6366–6375. doi: 10.1021/bi00268a008. [DOI] [PubMed] [Google Scholar]
  21. van Iersel J., Jzn J. F., Duine J. A. Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection. Anal Biochem. 1985 Nov 15;151(1):196–204. doi: 10.1016/0003-2697(85)90072-7. [DOI] [PubMed] [Google Scholar]

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