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Two distinct azurins function in the electron-transport chain of the obligate methylotroph Methylomonas J.

ABSTRACT: Methylomonas J is an obligate methylotroph although it is unable to grow on methane. Like Pseudomonas AM1, it produces two blue copper proteins when growing on methylamine, one of which is the recipient of electrons from the methylamine dehydrogenase. When grown on methanol, only the other blue copper protein is produced. We have determined the amino acid sequences of these blue copper proteins, and show that they are both true azurins. The sequences are clearly homologous to those of the proteins characterized from fluorescent pseudomonads and various species of Alcaligenes, and can be aligned with them and with each other without the need to postulate any internal insertions or deletions in the sequences. The iso-1 azurin, the one produced during both methanol and methylamine growth, shows 59-65% identity with these other azurins, whereas the iso-2 protein shows only 47-53% identity. The proteins show 52% identity with each other. The two functionally equivalent blue copper proteins from Pseudomonas AM1 belong to two sequence classes that are quite distinct from the true azurins. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication SUP 50151 (23 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1989) 257, 5.

SUBMITTER: Ambler RP

PROVIDER: S-EPMC1138853 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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Project description:The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O(2)-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J.192, 421-427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with alpha-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b(563) in methanol-limited bacteria. A third b-type cytochrome with an alpha-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a(3), which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O(2) in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

Project description:The mitochondrial electron transport chain (ETC) of Plasmodium malaria parasites is a major antimalarial drug target, but critical cytochrome functions remain unstudied and enigmatic. Parasites express two distinct cyt c homologs ( c and c -2) with unusually sparse sequence identity and uncertain fitness contributions. P. falciparum cyt c -2 is the most divergent eukaryotic cyt c homolog currently known and has sequence features predicted to be incompatible with canonical ETC function. We tagged both cyt c homologs and the related cyt c 1 for inducible knockdown. Translational repression of cyt c and cyt c 1 was lethal to parasites, which died from ETC dysfunction and impaired ubiquinone recycling. In contrast, cyt c -2 knockdown or knock-out had little impact on blood-stage growth, indicating that parasites rely fully on the more conserved cyt c for ETC function. Biochemical and structural studies revealed that both cyt c and c -2 are hemylated by holocytochrome c synthase, but UV-vis absorbance and EPR spectra strongly suggest that cyt c -2 has an unusually open active site in which heme is stably coordinated by only a single axial amino-acid ligand and can bind exogenous small molecules. These studies provide a direct dissection of cytochrome functions in the ETC of malaria parasites and identify a highly divergent Plasmodium cytochrome c with molecular adaptations that defy a conserved role in eukaryotic evolution.Significance statementMitochondria are critical organelles in eukaryotic cells that drive oxidative metabolism. The mitochondrion of Plasmodium malaria parasites is a major drug target that has many differences from human cells and remains poorly studied. One key difference from humans is that malaria parasites express two cytochrome c proteins that differ significantly from each other and play untested and uncertain roles in the mitochondrial electron transport chain (ETC). Our study revealed that one cyt c is essential for ETC function and parasite viability while the second, more divergent protein has unusual structural and biochemical properties and is not required for growth of blood-stage parasites. This work elucidates key biochemical properties and evolutionary differences in the mitochondrial ETC of malaria parasites.

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Two distinct azurins function in the electron-transport chain of the obligate methylotroph Methylomonas J.

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