化学生物学实验技术

Steady-state assays of nitrogenases share at least five requirements: an anaerobic environment, a consistent source of magnesium adenosine triphosphate (MgATP), a suitable source of reductant, a buffer system compatible with the product-quantification protocol to be used, and t ...

Nitrogenase is one of the most complex enzymes known to date. The extensively studied molybdenum nitrogenase consists of two protein components and three metal centers that are critical for nitrogenase activity. The inherent complexity of this enzyme system, which is further compoun ...

Biological nitrogen fixation is a complex and tightly regulated process limited to a group of prokaryotic species known as diazotrophs. Among well-studied diazotrophs, Azotobacter vinelandii is the best studied for its convenience of aerobic growth, its high levels of nitrogenase e ...

Nitrogenase-like dark operative protochlorophyllide oxidoreductase (DPOR) is involved in the two-electron reduction of protochlorophyllide to form chlorophyllide during chlorophyll biosynthesis. Formation of bacteriochlorophyll additionally requires a st ...

Advances in sequencing technology in the past decade have enabled the sequencing of genomes of thousands of organisms including diazotrophs. Genomics have enabled thorough analysis of the gene organization of nitrogen-fixing species, the identification of new genes involved in n ...

Biosynthesis of MoFe protein and, particularly, that of its associated P-cluster and FeMoco has raised a significant amount of interest because of the biological importance and chemical exclusiveness of these unique clusters. Following a brief introduction to the properties of Azot ...

Nitrogenase is the enzyme responsible for biological reduction of dinitrogen (N2) to ammonia, a form usable for life. Playing a central role in the global biogeochemical nitrogen cycle, this enzyme has been the focus of intensive research for over 60 years. This chapter provides an overview of t ...

The history of nitrogenase research dates all the way back to the 1800s. This chapter provides a brief account of the advances in this particular research area over the past few hundred years, which include such events as the initial discovery of biological nitrogen fixation, the preparation of a ...

X-ray absorption spectroscopy (XAS) involves the excitation of core electrons to bound states localized on the photoabsorber and the eventual excitation of the photoelectron to the continuum. The resulting spectra are typically divided into two regions: (1) the edge region which prov ...

Conformational changes imposed upon the Fe protein during binding and hydrolysis of Mg�ATP are key to initiating the cycle of interactions within the nitrogenase complex that result in gated electron transfer and the eventual multiple electron reduction of dinitrogen to ammonia. Won ...

EPR spectroscopy has been an important tool in nitrogenase research for the last 50 years. The three metalloclusters in nitrogenase, the Fe protein cluster, and the MoFe protein P-cluster, and FeMo-cofactor, all have EPR spectra when poised in the appropriate paramagnetic states. EPR spect ...

The iron-molybdenum cofactor (FeMoco) of the nitrogenase MoFe protein has remained a focal point in the field of bioinorganic chemistry for decades. This unique metal cluster has long been regarded as the actual site of dinitrogen reduction, and it is structurally complex and chemically un ...

M�ssbauer spectroscopy has contributed significantly to the studies of Fe-containing proteins. Early applications yielded detailed electronic characterizations of hemeproteins, and thus enhanced our understanding of the chemical properties of this important class of p ...

Being able to probe the structure and energy levels of metal ions in biological systems is an important goal of bioinorganic scientists. Several of the techniques used rely on the paramagnetic property of certain oxidation states of metal ions. MCD spectroscopy is one of those techniques and re ...

Synthetic compounds play an important role in developing our understanding of nitrogenase enzymes, and over the years, a multitude of new metal-containing compounds have been created using nitrogenase as an inspiration. The techniques for handling coordination compounds in orga ...

Modern density functional theory has tremendous potential with matching popularity in metalloenzymology to reveal the unseen atomic and molecular details of structural data, spectroscopic measurements, and biochemical experiments by providing insights into unobserv ...

Density functional theory (DFT) represents a unified framework for gaining molecular level insight into molybdenum–iron (MoFe) nitrogenase. However, accurately describing the electronic structure of the spin-polarized and spin-coupled iron–molybdenum cofactor (FeM ...

Genetic manipulation of the mouse has simplified the study of mammalian lipases and phospholipases at the level of whole animal physiology. Many insights into lipase biology derived from reductionist systems such as cultured cells have been confirmed using induced mutations in mice. B ...

LPL is regulated post-transcriptionally in response to several hormones. Post-translational regulation occurs in response to feeding (1,2). Glycosylation of LPL at the first N-linked glycosylation site is essential for catalytic activity and secretion (3). A number of studies have ...

This chapter describes the measurement of lipoprotein lipase (LPL) mRNA levels in adipose tissue and muscle. Many investigators measure LPL in human biopsy samples, where only a small amount of tissue is available, requiring sensitive measurement techniques. In addition, the descrip ...