Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

2021 ◽  
Author(s):  
James Birrell ◽  
Chris Furlan ◽  
Nipa Chongdar ◽  
Pooja Gupta ◽  
Wolfgang Lubitz ◽  
...  
Keyword(s):  
Active Sites ◽  
Structural Information ◽  
Thermotoga Maritima ◽  
Proton Reduction ◽  
Iron Sulfur Cluster ◽  
Electron Transfer Pathway ◽  
Iron Sulfur ◽  
Domain Movement ◽  
Cluster A ◽  
Structural Insight

Abstract Electron-bifurcation is a fundamental energy conservation mechanism in nature. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic primarily due to the lack of structural information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure is a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC heterotrimers electrically connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and proton reduction. Symmetry expansion identified two conformations of a flexible iron-sulfur cluster domain: a “closed bridge” and an “open bridge” conformation, where a Zn2+ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose two new mechanisms of electron-bifurcation in HydABC.

scholarly journals Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase

2021 ◽  
Author(s):  
Chris Furlan ◽  
Nipa Chongdar ◽  
Pooja Gupta ◽  
Wolfgang Lubitz ◽  
Hideaki Ogata ◽  
...  
Keyword(s):  
Energy Conservation ◽  
Structural Information ◽  
Thermotoga Maritima ◽  
Strongly Interacting ◽  
Domain Movement ◽  
Structural Insight

Electron-bifurcation is a fundamental energy conservation mechanism in nature. The electron-bifurcating [FeFe] hydrogenase from Thermotoga maritima (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron-bifurcation in HydABC remains enigmatic primarily due to the lack of structural information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure is a heterododecamer composed of two independent ‘halves’ each made of two strongly interacting HydABC trimers electrically connected via a [4Fe-4S] cluster, forming a bus-bar system. Symmetry expansion identified two conformations: a “closed bridge” and an “open bridge” conformation, where a Zn2+ site may act as a “hinge” allowing domain movement. Based on these structural revelations, we propose two new mechanisms of electron-bifurcation in HydABC.

scholarly journals Distinct Roles of the Salmonella enterica Serovar Typhimurium CyaY and YggX Proteins in the Biosynthesis and Repair of Iron-Sulfur Clusters

2014 ◽  
Vol 82 (4) ◽  
pp. 1390-1401 ◽  
Author(s):  
Jyoti Velayudhan ◽  
Joyce E. Karlinsey ◽  
Elaine R. Frawley ◽  
Lynne A. Becker ◽  
Margaret Nartea ◽  
...  
Keyword(s):  
Hydrogen Peroxide ◽  
Salmonella Enterica ◽  
Active Sites ◽  
Nitrosative Stress ◽  
Iron Sulfur Cluster ◽  
Iron Sulfur Clusters ◽  
Content Type ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Serovar Typhimurium

ABSTRACTLabile [4Fe-4S]2+clusters found at the active sites of many dehydratases are susceptible to damage by univalent oxidants that convert the clusters to an inactive [3Fe-4S]1+form. Bacteria repair damaged clusters in a process that does not requirede novoprotein synthesis or the Isc and Suf cluster assembly pathways. The current study investigates the participation of the bacterial frataxin ortholog CyaY and the YggX protein, which are proposed to play roles in iron trafficking and iron-sulfur cluster repair. Previous reports found that individual mutations incyaYoryggXwere not associated with phenotypic changes inEscherichia coliandSalmonella entericaserovar Typhimurium, suggesting that CyaY and YggX might have functionally redundant roles. However, we have found that individual mutations incyaYoryggXconfer enhanced susceptibility to hydrogen peroxide inSalmonella entericaserovar Typhimurium. In addition, inactivation of thestm3944open reading frame, which is located immediately upstream ofcyaYand which encodes a putative inner membrane protein, dramatically enhances the hydrogen peroxide sensitivity of acyaYmutant. Overexpression of STM3944 reduces the elevated intracellular free iron levels observed in anS. Typhimuriumfurmutant and also reduces the total cellular iron content under conditions of iron overload, suggesting that thestm3944-encoded protein may mediate iron efflux. Mutations incyaYandyggXhave different effects on the activities of the iron-sulfur cluster-containing aconitase, serine deaminase, and NADH dehydrogenase I enzymes ofS. Typhimurium under basal conditions or following recovery from oxidative stress. In addition,cyaYandyggXmutations have additive effects on 6-phosphogluconate dehydratase-dependent growth during nitrosative stress, and acyaYmutation reducesSalmonellavirulence in mice. Collectively, these results indicate that CyaY and YggX play distinct supporting roles in iron-sulfur cluster biosynthesis and the repair of labile clusters damaged by univalent oxidants.Salmonellaexperiences oxidative and nitrosative stress within host phagocytes, and CyaY-dependent maintenance of labile iron-sulfur clusters appears to be important forSalmonellavirulence.

scholarly journals Recombination of 2Fe-2S ferredoxins reveals differences in the inheritance of thermostability and midpoint potential

2020 ◽  
Author(s):  
Ian J. Campbell ◽  
Dimithree Kahanda ◽  
Joshua T. Atkinson ◽  
Othneil N. Sparks ◽  
Jinyoung Kim ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Physical Properties ◽  
Sulfite Reductase ◽  
Amino Acid Substitutions ◽  
Iron Sulfur Cluster ◽  
Midpoint Potential ◽  
Electron Transfer Pathway ◽  
Iron Sulfur ◽  
Wide Range ◽  
Electron Carriers

ABSTRACTHomologous recombination can be used to create enzymes that exhibit distinct activities and stabilities from proteins in nature, allowing researchers to overcome component limitations in synthetic biology. To investigate how recombination affects the physical properties of an oxidoreductase that transfers electrons, we created ferredoxin (Fd) chimeras by recombining distantly-related cyanobacterial and cyanomyophage Fds that present similar midpoint potentials but distinct thermostabilities. Fd chimeras having a wide range of amino acid substitutions retained the ability to coordinate an iron-sulfur cluster, although their thermostabilities varied with the fraction of residues inherited from each parent. The midpoint potentials of chimeric Fds also varied. However, all of the synthetic Fds exhibited midpoint potentials outside of the parental protein range. Each of the chimeric Fds could also be used to build synthetic pathways that support electron transfer between Fd-NADP reductase and sulfite reductase in Escherichia coli, although the chimeric Fds varied in the expression required to support similar levels of cellular electron transfer. These results show how recombination can be used to rapidly diversify the physical properties of protein electron carriers and reveal differences in the inheritance of thermostability and electrochemical properties. Furthermore, they illustrate how electron transfer efficiencies of chimeric Fds can be rapidly evaluated using a synthetic electron transfer pathway.

scholarly journals Structural properties of [2Fe-2S] ISCA2-IBA57: a complex of the mitochondrial iron-sulfur cluster assembly machinery

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Veronica Nasta ◽  
Stefano Da Vela ◽  
Spyridon Gourdoupis ◽  
Simone Ciofi-Baffoni ◽  
Dmitri I. Svergun ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Information ◽  
Specific Interaction ◽  
Integrative Approach ◽  
Cluster Assembly ◽  
Experimental Conditions ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Complex Protein

AbstractIn mitochondria, a complex protein machinery is devoted to the maturation of iron-sulfur cluster proteins. Structural information on the last steps of the machinery, which involve ISCA1, ISCA2 and IBA57 proteins, needs to be acquired in order to define how these proteins cooperate each other. We report here the use of an integrative approach, utilizing information from small-angle X-ray scattering (SAXS) and bioinformatics-driven docking prediction, to determine a low-resolution structural model of the human mitochondrial [2Fe-2S]2+ ISCA2-IBA57 complex. In the applied experimental conditions, all the data converge to a structural organization of dimer of dimers for the [2Fe-2S]2+ ISCA2-IBA57 complex with ISCA2 providing the homodimerization core interface. The [2Fe-2S] cluster is out of the ISCA2 core while being shared with IBA57 in the dimer. The specific interaction pattern identified from the dimeric [2Fe-2S]2+ ISCA2-IBA57 structural model allowed us to define the molecular grounds of the pathogenic Arg146Trp mutation of IBA57. This finding suggests that the dimeric [2Fe-2S] ISCA2-IBA57 hetero-complex is a physiologically relevant species playing a role in mitochondrial [4Fe-4S] protein biogenesis.

scholarly journals Glutathione-coordinated [2Fe–2S] cluster: a viable physiological substrate for mitochondrial ABCB7 transport

2015 ◽  
Vol 51 (12) ◽  
pp. 2253-2255 ◽  
Author(s):  
Jingwei Li ◽  
J. A. Cowan
Keyword(s):  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Mitochondrial Iron ◽  
Cluster A ◽  
Export Protein

Glutathione-coordinated [2Fe–2S] cluster is demonstrated to be a viable and likely substrate for mitochondrial iron–sulfur cluster transport by the ABCB7 export protein.

Structural Insight into Poplar Glutaredoxin C1 with a Bridging Iron−Sulfur Cluster at the Active Site†,‡

Biochemistry ◽  
2006 ◽  
Vol 45 (26) ◽  
pp. 7998-8008 ◽  
Author(s):  
Yingang Feng ◽  
Nan Zhong ◽  
Nicolas Rouhier ◽  
Toshiharu Hase ◽  
Masami Kusunoki ◽  
...  
Keyword(s):  
Active Site ◽  
Iron Sulfur Cluster ◽  
Sulfur Cluster ◽  
Iron Sulfur ◽  
Structural Insight ◽  
Insight Into

scholarly journals The periplasmic nitrate reductase in Shewanella: the resolution, distribution and functional implications of two NAP isoforms, NapEDABC and NapDAGHB

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 302-312 ◽  
Author(s):  
Philippa J. L. Simpson ◽  
David J. Richardson ◽  
Rachel Codd
Keyword(s):  
Nitrate Reductase ◽  
Shewanella Oneidensis ◽  
Unusual Feature ◽  
Iron Sulfur Cluster ◽  
Protein Subunits ◽  
Sulfur Cluster ◽  
Periplasmic Nitrate Reductase ◽  
Iron Sulfur ◽  
Functional Implications ◽  
Cluster A

In the bacterial periplasm, the reduction of nitrate to nitrite is catalysed by a periplasmic nitrate reductase (NAP) system, which is a species-dependent assembly of protein subunits encoded by the nap operon. The reduction of nitrate catalysed by NAP takes place in the 90 kDa NapA subunit, which contains a Mo-bis-molybdopterin guanine dinucleotide cofactor and one [4Fe−4S] iron–sulfur cluster. A review of the nap operons in the genomes of 19 strains of Shewanella shows that most genomes contain two nap operons. This is an unusual feature of this genus. The two NAP isoforms each comprise three isoform-specific subunits – NapA, a di-haem cytochrome NapB, and a maturation chaperone NapD – but have different membrane-intrinsic subunits, and have been named NAP-α (NapEDABC) and NAP-β (NapDAGHB). Sixteen Shewanella genomes encode both NAP-α and NAP-β. The genome of the vigorous denitrifier Shewanella denitrificans OS217 encodes only NAP-α and the genome of the respiratory nitrate ammonifier Shewanella oneidensis MR-1 encodes only NAP-β. This raises the possibility that NAP-α and NAP-β are associated with physiologically distinct processes in the environmentally adaptable genus Shewanella.

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