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Abstract Background The bioenergetics of Archaea with respect to the evolution of electron transfer systems is very interesting. In contrast to terminal oxidases, a canonical bc1 complex has not yet been isolated from Archaea. In particular, c-type cytochromes have been reported only for a limited number of species. Results Here, we isolated a c-type cytochrome-containing enzyme complex from the membranes of the hyperthermophilic archaeon, Aeropyrum pernix, grown aerobically. The redox spectrum of the isolated c-type cytochrome showed a characteristic α-band peak at 553 nm corresponding to heme C. The pyridine hemochrome spectrum also revealed the presence of heme B. In non-denaturing polyacrylamide gel electrophoresis, the cytochrome migrated as a single band with an apparent molecular mass of 80 kDa, and successive SDS-PAGE separated the 80-kDa band into 3 polypeptides with apparent molecular masses of 40, 30, and 25 kDa. The results of mass spectrometry indicated that the 25-kDa band corresponded to the hypothetical cytochrome c subunit encoded by the ORF APE_1719.1. In addition, the c-type cytochrome-containing polypeptide complex exhibited menaquinone: yeast cytochrome c oxidoreductase activities. Conclusion In conclusion, we showed that A. pernix, a hyperthemophilic archaeon, has a "full" bc complex that includes a c-type cytochrome, and to the best of our knowledge, A. pernix is the first archaea from which such a bc complex has been identified. However, an electron donor candidates for cytochrome c oxidase, such as a blue copper protein, have not yet been identified in the whole genome data of this archaeon. We are currently trying to identify an authentic substrate between a bc complex and terminal oxidase.

Cytochromes bd are essential for microaerobic respiration of many prokaryotes including a number of human pathogens. These enzymes catalyze the reduction of molecular oxygen to water using quinols as electron donors. Their importance for prokaryotic survival and the absence of eukaryotic homologs make these enzyme ideal targets for antimicrobial drugs. Here, we determined the cryoEM structure of the menaquinol-oxidizing cytochrome bd -type oxygen reductase of the facultative anaerobic Actinobacterium Corynebacterium glutamicum at a resolution of 2.7 Å. The obtained structure adopts the signature pseudosymmetrical heterodimeric architecture of canonical cytochrome bd oxidases formed by the core subunits CydA and CydB. No accessory subunits were identified for this cytochrome bd homolog. The two b -type hemes and the oxygen binding heme d are organized in a triangular geometry with a protein environment around these redox cofactors similar to that of the closely related cytochrome bd from M. tuberculosis . We identified oxygen and a proton conducting channels emerging from the membrane space and the cytoplasm, respectively. Compared to the prototypical enzyme homolog from the E. coli , the most apparent difference is found in the location and size of the proton channel entry site. In canonical cytochrome bd oxidases quinol oxidation occurs at the highly flexible periplasmic Q-loop located in the loop region between TMHs six and seven. An alternative quinol-binding site near heme b 595 was previously identified for cytochrome bd from M. tuberculosis . We discuss the relevance of the two quinol oxidation sites in actinobacterial bd -type oxidases and highlight important differences that may explain functional and electrochemical differences between C. glutamicum and M. tuberculosis . This study expands our current understanding of the structural diversity of actinobacterial and proteobacterial cytochrome bd oxygen reductases and provides deeper insights into the unique structural and functional properties of various cytochrome bd variants from different phylae., (Copyright © 2023 Grund, Kabashima, Kusumoto, Wu, Welsch, Sakamoto, Michel and Safarian.)

We address the problem of evaluating the transfer entropy (TE) produced by biochemical reactions from experimentally measured data. Although these reactions are generally nonlinear and nonstationary processes making it challenging to achieve accurate modeling, Gaussian approximation can facilitate the TE assessment only by estimating covariance matrices using multiple data obtained from simultaneously measured time series representing the activation levels of biomolecules such as proteins. Nevertheless, the nonstationary nature of biochemical signals makes it difficult to theoretically assess the sampling distributions of TE, which are necessary for evaluating the statistical confidence and significance of the data-driven estimates. We resolve this difficulty by computationally assessing the sampling distributions using techniques from computational statistics. The computational methods are tested by using them in analyzing data generated from a theoretically tractable time-varying signal model, which leads to the development of a method to screen only statistically significant estimates. The usefulness of the developed method is examined by applying it to real biological data experimentally measured from the ERBB-RAS-MAPK system that superintends diverse cell fate decisions. A comparison between cells containing wild-type and mutant proteins exhibits a distinct difference in the time evolution of TE while any apparent difference is hardly found in average profiles of the raw signals. Such a comparison may help in unveiling important pathways of biochemical reactions.