Three multihaem cytochromes c from the hyperthermophilic archaeon Ignicoccus hospitalis: purification, properties and localization

Abstract

Three different multihaem cytochromes c were purified from cell extracts of the hyperthermophilic archaeon Ignicoccus hospitalis. One tetrahaem cytochrome, locus tag designation Igni_0530, was purified from membrane fractions together with the iron–sulfur protein Igni_0529. Two octahaem cytochromes, Igni_0955 and Igni_1359, were purified from soluble fractions but were also present in the membrane fraction. N-terminal sequencing showed that three of the four proteins had their signal peptides cleaved off, while results were ambiguous for Igni_0955. In contrast, mass spectrometry of Igni_0955 and Igni_1359 resulted in single mass peaks including the signal sequences and eight haems per subunit and so both forms might be present in the cell. Igni_0955 and Igni_1359 belong to the hydroxylamine dehydrogenase (HAO) family (29% mutual identity). HAO or reductase activities with inorganic sulfur compounds were not detected. Igni_0955 was reduced by enriched I. hospitalis hydrogenase at a specific activity of 243 nmol min^-1 (mg hydrogenase)^-1 while activity was non-existent for Igni_0530 and low for Igni_1359. Immuno-electron microscopy of ultra-thin sections showed that Igni_0955 and Igni_1359 are located in both I. hospitalis membranes and also in the intermembrane compartment. We concluded that these cytochromes might function as electron shuttles between the hydrogenase in the outer cellular membrane and cellular reductases, whereas Igni_0530 might be part of the sulfur-reducing mechanism

Cytochromes c in Archaea: distribution, maturation, cell architecture, and the special case of Ignicoccus hospitalis

“…We had purified three different Cytc from I . hospitalis cells, however, we can so far only speculate about their in vivo function (Naß et al, 2014 ). Two of these proteins, named Igni_0955 and Igni_1359 after their GenBank locus tag numbers, were present in soluble and membrane extracts, while the third one (Igni_0530) was present only in the membrane fractions.…”

“…Apart from I . hospitalis (Naß et al, 2014 ), Cytc were found biochemically in the hydrogen-oxidizing and sulfur-reducing complex of the related Archaea Pyrodictium abyssi and P . brockii (Pihl et al, 1992 ; Dirmeier et al, 1998 ), in a bc 1 complex from the likewise related microaerophilic Aeropyrum pernix (Kabashima and Sakamoto, 2011 ), in the nitrate reducer Pyrobaculum aerophilum (Feinberg and Holden, 2006 ; all of them hyperthermophilic Crenarchaeota ), in cultured ( Methanosarcina spp.)…”

“…Surprisingly, experimental gene identification was accomplished only for a few of these species including the three multiheme Cytc from I. hospitalis and of the bc 1 complex of the related A . pernix (Kabashima and Sakamoto, 2011 ; Naß et al, 2014 ).…”

ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus

“…Once on the cell surface, a subset of secreted proteins are released into the extracellular environment. However, the vast majority are anchored to the cell surface, i.e., to the cytoplasmic membrane of archaea and Gram-positive bacteria, to the inner (cytoplasmic) membrane or the outer (surface) membrane of Gram-negative bacteria and a subset of archaea, or to the prokaryotic cell wall (7,(9)(10)(11). A diverse array of protein-anchoring pathways have evolved to facilitate retention of these secreted proteins at the cell surface, including a recently described mechanism that facilitates C-terminal lipid-mediated anchoring of prokaryotic proteins (12)(13)(14).…”

Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics of Ignicoccus Species

“…With the findings that ATP synthase and sulfur-hydrogen oxidoreductase (27), as well as acetyl-CoA synthetase (36), are located in/associated with the OCM, it became clear that several essential cellular processes are located in the IMC. Further studies indicate that cytochromes are likely involved in electron transport within the OCM (37). However, there is not much information about the chemical composition (e.g., predominant ions and pH) in the IMC.…”