权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Biosynthesis and Reactivity of the Active Site of the [FeFe]-Hydrogenases

[FeFe]-氢化酶活性位点的生物合成和反应性

基本信息

批准号：
9912778
负责人：
Alison R Fout
金额：
$ 30.87万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-07-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9912778
关键词：
Acidity Active Sites Affect Amines Ammonium Anabolism Antidotes Archaea Bacteria Behavior Binding Biochemistry Chemicals Collaborations Complex Consensus Coupled Crystallization Cyanides Cysteine Development Electrostatics Enzymes Exhibits Formaldehyde Heavy Metals Human Hydrogenase In Vitro Iron Isotope Labeling Ligands Mediating Methods Modeling Organism Organometallic Chemistry Oxidation-Reduction Pathogenicity Pharmacologic Substance Property Proteins Protons Reaction Reagent Role Route Rubredoxins Scaffolding Protein Site Source Spectrum Analysis Structure Synchrotrons Testing Water Work base biophysical analysis biophysical techniques catalyst chemical reaction cofactor experimental study in vitro Assay in vitro testing inhibitor/antagonist interest metal poisoning nickel-iron hydrogenase novel pathogen protonation scaffold skills theories

项目摘要

Hydrogenase enzymes are pervasive, being found in bacteria, archaea, and some higher organisms. Hydrogenases are found in many pathogens, including some that inhabit the human gut. The [FeFe] and [NiFe]-hydrogenases mediate the most fundamental chemical reaction: the interconversion of H2 with protons and reducing equivalents. Since H2 is an unusual substrate, the enzymes are also structurally exceptional with an array of distinctive cofactors, especially the site of H2 binding and release. Similarly, the biosynthesis of these active sites involves elaborate and novel biochemistry. In addition to biophysical approaches, elucidation of these mechanisms relies on organometallic chemistry, especially since the substrates (H2, H+, H-) are often invisible to conventional biophysical methods. This project aims to elucidate the biosynthesis and mechanism of action of the [FeFe] enzymes, the faster of the two main hydrogenases and the one most amenable to development for other applications. The work involves synthesis of proposed intermediates, spectroscopic and electrochemical characterization, in vitro assays, and isotopic labeling. It also relies on extensive collaborations with groups that offer specialized skills in theory, synchrotron-based spectroscopy, spin resonance, and in vitro testing. This project specifically focuses on the recently confirmed azadithiolate (adt) cofactor, which is the enzyme's most remarkable component from a mechanistic perspective. Although the adt cofactor is unstable in the free state, methods are being developed to stabilize it in protected form. It will be isotopically labeled and incorporated into an apo enzyme. These experiments will allow us to identify the precursor to the adt (main hypothesis: radical SAM induced reactions of cysteine-Fe conjugates). Prior to construction of the Fe2(adt) ensemble, two Fe-cysteine-CO centers are supposed to be preorganized in a scaffold protein HydF. This work will produce the first Fe-cysteine-CO complexes for testing in vitro and by chemical methods. Finally, the work will evaluate the role of adt on the catalytic mechanism. Of specific interest is the influence of the protonation state of the amine cofactor on the behavior of the [2Fe] subunit. The latter studies will be conducted on model complexes containing Fe2 with authentic and modified amine cofactors. Of specific interest is the influence of N-protonation on the binding of H2 and the inhibitors CO and formaldehyde. Overall, the work focuses on understanding the biosynthesis and mechanism of action of the fastest catalysts known for producing H2 from water.

氢化酶无处不在，存在于细菌、古生菌和一些高等生物中。