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Monitoring H-cluster assembly using a semi-synthetic HydF protein
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Molekylär biomimetik.ORCID-id: 0000-0002-6717-6612
2019 (engelsk)Inngår i: Dalton Transactions, ISSN 1477-9226, E-ISSN 1477-9234, Vol. 48, nr 18, s. 5978-5986Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

The [FeFe] hydrogenase enzyme interconverts protons and molecular hydrogen with remarkable efficiency. The reaction is catalysed by a unique metallo-cofactor denoted as the H-cluster containing an organometallic dinuclear Fe component, the [2Fe] subsite. The HydF protein delivers a precursor of the [2Fe] subsite to the apo-[FeFe] hydrogenase, thus completing the H-cluster and activating the enzyme. Herein we generate a semi-synthetic form of HydF by loading it with a synthetic low valent dinuclear Fe complex. We show that this semi-synthetic protein is practically indistinguishable from the native protein, and utilize this form of HydF to explore the mechanism of H-cluster assembly. More specifically, we show that transfer of the precatalyst from HydF to the hydrogenase enzyme results in the release of CO, underscoring that the pre-catalyst is a four CO species when bound to HydF. Moreover, we propose that an electron transfer reaction occurs during H-cluster assembly, resulting in an oxidation of the [2Fe] subsite with concomitant reduction of the [4Fe4S] cluster present on the HydF protein.

sted, utgiver, år, opplag, sider
ROYAL SOC CHEMISTRY , 2019. Vol. 48, nr 18, s. 5978-5986
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-390520DOI: 10.1039/c8dt04294bISI: 000472449300013PubMedID: 30632592OAI: oai:DiVA.org:uu-390520DiVA, id: diva2:1342710
Forskningsfinansiär
Swedish Research Council, 621-2014-5670Swedish Research Council Formas, 213-2014-880EU, European Research Council, 714102Tilgjengelig fra: 2019-08-14 Laget: 2019-08-14 Sist oppdatert: 2019-09-18bibliografisk kontrollert
Inngår i avhandling
1. The birth of the hydrogenase: Studying the mechanism of [FeFe] hydrogenase maturation
Åpne denne publikasjonen i ny fane eller vindu >>The birth of the hydrogenase: Studying the mechanism of [FeFe] hydrogenase maturation
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

The [FeFe] hydrogenases are ancient metalloenzymes that catalyse the reversible interconversion between protons, electrons and molecular hydrogen. Despite the large structural variability within the [FeFe] hydrogenase family, the active site, the so called “H-cluster” is present in every representative. The H-cluster is composed by a four cysteine coordinated [4Fe4S] cluster, ligated via a shared cysteine to a biologically unique [2Fe] subsite decorated with CO and CN ligands and an azadithiolate bridging ligand. The biosynthesis of the [2Fe] subsite requires a maturation machinery, composed of at least three maturase enzymes, denoted HydG, HydE, and HydF. HydE and HydG are members of the radical SAM enzyme family, and are responsible for the construction of a pre-catalyst on HydF. This pre-catalyst is finally transferred from HydF to HydA, where it becomes part of the H-cluster.

Recently, a pioneer study combined synthetic chemistry and biochemistry in order to create semi-synthetic HydF proteins. Synthetic mimics of the [2Fe] subsite were introduced to HydF, and this resulting semi-synthetic HydF was used to activate the unmatured hydrogenase (apo-HydA). This technique ushered in a new era in [FeFe] hydrogenase research.

This thesis work is devoted to a deeper understanding of H-cluster formation and [FeFe] hydrogenase maturation, and this process is studied using standard molecular biological and biochemical techniques, and EPR, FTIR, XAS and GEMMA spectroscopic techniques combined with this new type of chemistry mentioned above. EPR spectroscopy was employed to verify the construction of a semi-synthetic [FeFe] hydrogenase inside living cells. The addition of a synthetic complex to cell cultures expressing apo-HydA resulted in a rhombic EPR signal, attributable to an Hox-like species. Moreover, the assembly mechanism of the H-cluster was probed in vitro using XAS, EPR, and FTIR spectroscopy. We verified with all three techniques that the Hox-CO state is formed on a time-scale of seconds, and this state slowly turns into the catalytically active Hox via release of a CO ligand. Furthermore, a semi-synthetic form of the HydF protein from Clostridium acetobutylicum was prepared and characterized in order to prove that such semi-synthetic forms of HydF are biologically relevant. Finally,GEMMA measurements were performed to elucidate the quaternary structure of the HydF-HydA interaction, revealing that dimeric HydF is interacting with a monomeric HydA. However, mutant HydF proteins were prepared, lacking the dimerization (as well as its GTPase) domain, and these severely truncated forms of HydF was found to still retain the capacity to both harbor the pre-catalyst as well as transferring it to apo-HydA. These observations highlight the multi-functionality of HydF, where different domains are critical in different steps of the maturation, that is the dimerization and GTPase domain are rather involved in pre-catalyst assembly rather than its transfer to apo-HydA.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 78
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1856
Emneord
HydF, HydA, [FeFe] hydrogenase maturation, semi-synthetic enzymes
HSV kategori
Forskningsprogram
Biokemi
Identifikatorer
urn:nbn:se:uu:diva-393261 (URN)978-91-513-0754-1 (ISBN)
Disputas
2019-11-06, Å4101, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Forskningsfinansiär
Swedish Research Council, 62120145670Swedish Research Council Formas, 2132014880
Tilgjengelig fra: 2019-10-11 Laget: 2019-09-18 Sist oppdatert: 2019-11-12

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