Dataset Information

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

ABSTRACT: Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.

SUBMITTER: Dijkstra M

PROVIDER: S-EPMC1149026 | biostudies-other | 1988 Apr

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Previous studies demonstrated that an outer membrane c-type cytochrome, OmcB (GSU_2737), was involved in Fe(III) reduction in Geobacter sulfurreducens. An OmcB-deficient mutant was greatly impaired in its ability to reduce both soluble and insoluble Fe(III). Reintroducing omcB restored the capacity for Fe(III) reduction at a level proportional to the level of OmcB production. Here, we report that the OmcB-deficient mutant gradually adapted to grow on soluble Fe(III) but not insoluble Fe(III). The adapted OmcB-deficient mutant reduced soluble Fe(III) at a rate comparable to that of the wild type, but the cell yield of the mutant was only ca. 60% of that of the wild type under steady-state culturing conditions. Analysis of proteins and transcript levels demonstrated that expression of several membrane-associated cytochromes was higher in the adapted mutant than in the wild type. Further comparison of transcript levels during steady-state growth on Fe(III) citrate with a whole-genome DNA microarray revealed a significant shift in gene expression in an apparent attempt to adapt metabolism to the impaired electron transport to Fe(III). These results demonstrate that, although there are many other membrane-bound c-type cytochromes in G. sulfurreducens, increased expression of these cytochromes cannot completely compensate for the loss of OmcB. The concept that outer membrane cytochromes are promiscuous reductases that are interchangeable in function appears to be incorrect. Furthermore, the results indicate that there may be different mechanisms for electron transfer to soluble Fe(III) and insoluble Fe(III) oxides in G. sulfurreducens, which emphasizes the importance of studying electron transport to the environmentally relevant Fe(III) oxides. (From Leang C, Adams LA, Chin KJ, Nevin KP, Methé BA, Webster J, Sharma ML, Lovley DR. Adaptation to disruption of the electron transfer pathway for Fe(III) reduction in Geobacter sulfurreducens. J Bacteriol. 2005 Sep;187(17):5918-26.) Keywords: Geobacter, gene expression, genetic modification

Project description: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.

Dataset Information

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets