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

1989 ◽  
Vol 257 (1) ◽  
pp. 87-94 ◽  
Author(s):  
M Dijkstra ◽  
J Frank ◽  
J A Duine
Keyword(s):  
Electron Transfer ◽  
Electron Acceptor ◽  
Substrate Oxidation ◽  
Methanol Dehydrogenase ◽  
Methylotrophic Bacteria ◽  
Ph Optimum ◽  
Oxidation Step ◽  
Reaction Cycles ◽  
Rate Limiting

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.

scholarly journals The interaction of methanol dehydrogenase and its cytochrome electron acceptor

1995 ◽  
Vol 312 (1) ◽  
pp. 261-265 ◽  
Author(s):  
S L Dales ◽  
C Anthony
Keyword(s):  
Electron Transfer ◽  
Electron Acceptor ◽  
Methanol Dehydrogenase ◽  
Ionic Interactions ◽  
Optimal Position ◽  
Factors Affecting ◽  
Carboxylate Groups ◽  
Initial Interaction ◽  
Cytochrome Cl ◽  
Initial Binding

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial ‘docking’ of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the ‘docked’ cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.

scholarly journals MacA, a Diheme c-Type Cytochrome Involved in Fe(III) Reduction by Geobacter sulfurreducens

2004 ◽  
Vol 186 (12) ◽  
pp. 4042-4045 ◽  
Author(s):  
Jessica E. Butler ◽  
Franz Kaufmann ◽  
Maddalena V. Coppi ◽  
Cinthia Núñez ◽  
Derek R. Lovley
Keyword(s):  
Oxidative Stress ◽  
Electron Transfer ◽  
Electron Acceptor ◽  
Peroxidase Activity ◽  
Geobacter Sulfurreducens

ABSTRACT A 36-kDa diheme c-type cytochrome abundant in Fe(III)-respiring Geobacter sulfurreducens, designated MacA, was more highly expressed during growth with Fe(III) as the electron acceptor than with fumarate. Although MacA has homology to proteins with in vitro peroxidase activity, deletion of macA had no impact on response to oxidative stress. However, the capacity for Fe(III) reduction was greatly diminished, indicating that MacA, which is predicted to be localized in the periplasm, is a key intermediate in electron transfer to Fe(III).

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

1988 ◽  
Vol 251 (2) ◽  
pp. 467-474 ◽  
Author(s):  
M Dijkstra ◽  
J Frank ◽  
J E van Wielink ◽  
J A Duine
Keyword(s):  
Electron Acceptor ◽  
Protein Molecule ◽  
Methanol Dehydrogenase ◽  
Methylotrophic Bacteria ◽  
High Ph ◽  
Native Proteins ◽  
Cytochromes C ◽  
Reverse Situation ◽  
Cytochrome Cl ◽  
Reduced Forms

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.

scholarly journals Development of engineered ferredoxin reductase systems for the efficient hydroxylation of steroidal substrates

2020 ◽  
Author(s):  
Zhangliang Zhu ◽  
Xin Gao ◽  
Zhan Song ◽  
Chao Li ◽  
Fuping Lu ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Catalytic Efficiency ◽  
Nadh Regeneration ◽  
Whole Cell ◽  
Rate Limiting Step ◽  
Engineering Strategy ◽  
Whole Cell Catalysis ◽  
Rate Limiting ◽  
Insight Into

Abstract Background 9α-hydroxy-4-androstene-3,17-dione (9OHAD), catalyzed by 3-ketosteroid-9-hydroxylase (KSH) using 4-androstene-3,17-dione (AD) as a substrate, is an important precursor for the synthesis of adrenocortical hormones. Whole-cell catalyst in microorganisms with this KSH system for desirable hydroxylated steroids in high purity and productivity have rarely been successful. Results The rate-limiting step for the biosynthesis of 9OHAD is catalyzed by the reductase KshB, which is an important component of electrons donor. A sufficient supply system of the cofactor NADH was constructed on KshB by introducing the formate dehydrogenase (FDH) gene. Several reductases were then screened to find a TDO reductase containing a ferredoxin, which showed a maximal NADH activity with a catalytic efficiency (kcat/Km) of 0.43 s− 1 µM− 1 and 54.8% of 9OHAD yield via multienzyme cascade catalysis in vitro. TDO mutagenesis was further performed via a protein engineering strategy, resulting in a 2.25-fold improvement in activity and a 74.8% 9OHAD yield. The modification of a Rieske [2Fe-2S] cluster in KshB and TDO showed 9OHAD yields of 56.1% and 74.5% higher than wild-type ones, which implied Rieske [2Fe-2S] ferredoxin strengthening for electrons transferring. The biosynthesis of 9OHAD was further optimized in a whole-cell catalysis system with FDH, KshA, and TDO_M9 mutant with the Rieske [2Fe-2S] ferredoxin (BLKA-RMT-F), resulting in a final production of 5.24 g/L 9OHAD, a considerable yield of 99.3% of theoretical without by-products. Conclusion An efficient whole-cell catalyst was constructed with considerable production and yield by modification of a Rieske [2Fe-2S] cluster in TDO with increased the efficiency of electron transfer. This research provided comprehensive insight into the electron transfer system for these steroid hydroxylation reactions and NADH regeneration systems.

scholarly journals The three-dimensional structures of methanol dehydrogenase from two methylotrophic bacteria at 2.6-A resolution.

1992 ◽  
Vol 267 (31) ◽  
pp. 22289-22297
Author(s):  
Z.X. Xia ◽  
W.W. Dai ◽  
J.P. Xiong ◽  
Z.P. Hao ◽  
V.L. Davidson ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Methanol Dehydrogenase ◽  
Methylotrophic Bacteria

scholarly journals Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

1996 ◽  
Vol 271 (43) ◽  
pp. 27188
Author(s):  
Lloyd W. Ruddock ◽  
Jeremy J.F. Coen ◽  
Caroline Cheesman ◽  
Robert B. Freedman ◽  
Timothy R. Hirst
Keyword(s):  
Escherichia Coli ◽  
Molecular Basis ◽  
B Subunit ◽  
Heat Labile ◽  
Rate Limiting

scholarly journals Molecular Engineering for High-Performance Fullerene Broadband Photodetector

2021 ◽  
Author(s):  
Mingming Su ◽  
Yajing Hu ◽  
Ao Yu ◽  
Zhiyao Peng ◽  
Wangtao Long ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Low Temperature ◽  
Electron Acceptor ◽  
Photoinduced Electron Transfer ◽  
High Performance ◽  
Organic Molecules ◽  
Low Cost ◽  
Molecular Engineering ◽  
Temperature Requirement ◽  
High Flexibility

Broadband photodetectors fabricated with organic molecules have the advantages of low cost, high flexibility, easy processing and low-temperature requirement. Fullerene molecules, due to the electron acceptor and photoinduced electron transfer...

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