Probing conformational dynamics of DNA binding by CO-sensing transcription factor, CooA.

The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (μs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme.

Molecular Determinants of Efficient Cobalt-Substituted Hemoprotein Production in .

Exchanging the native iron of heme for other metals yields artificial metalloproteins with new properties for spectroscopic studies and biocatalysis. Recently, we reported a method for the biosynthesis and incorporation of a non-natural metallocofactor, cobalt protoporphyrin IX (CoPPIX), into hemoproteins using the common laboratory strain BL21(DE3). This discovery inspired us to explore the determinants of metal specificity for metallocofactor biosynthesis in .

Carbon Monoxide-Sensing Transcription Factors: Regulators of Microbial Carbon Monoxide Oxidation Pathway Gene Expression.

Carbon monoxide (CO) serves as a source of energy and carbon for a diverse set of microbes found in anaerobic and aerobic environments. The enzymes that bacteria and archaea use to oxidize CO depend upon complex metallocofactors that require accessory proteins for assembly and proper function. This complexity comes at a high energetic cost and necessitates strict regulation of CO metabolic pathways in facultative CO metabolizers to ensure that gene expression occurs only when CO concentrations and redox conditions are appropriate.

Redox Brake Regulator RedB and FnrL Function as Yin-Yang Regulators of Anaerobic-Aerobic Metabolism in Rhodobacter capsulatus.

We recently described a new member of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family called RedB, an acronym for redox brake, that functions to limit the production of ATP and NADH. This study shows that the RedB regulon significantly overlaps the FnrL regulon, with 199 genes being either directly or indirectly regulated by both of these global regulatory proteins. Among these 199 coregulated genes, 192 are divergently regulated, indicating that RedB functions as an antagonist of FnrL.

RedB, a Member of the CRP/FNR Family, Functions as a Transcriptional Redox Brake.

Phylogenetic and sequence similarity network analyses of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family of transcription factors indicate the presence of numerous subgroups, many of which have not been analyzed. Five homologs of the CRP/FNR family are present in the Rhodobacter capsulatus genome. One is a member of a broadly disseminated, previously uncharacterized CRP/FNR family subgroup encoded by the gene .

Quaternary Structure and Deoxyribonucleic Acid-Binding Properties of the Heme-Dependent, CO-Sensing Transcriptional Regulator RcoM.

RcoM, a heme-containing, CO-sensing transcription factor, is one of two known bacterial regulators of CO metabolism. Unlike its analogue CooA, the structure and DNA-binding properties of RcoM remain largely uncharacterized. Using a combination of size exclusion chromatography and sedimentation equilibrium, we demonstrate that RcoM-1 from is a dimer, wherein the heme-binding domain mediates dimerization.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in and incorporation into hemoproteins.

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions.

Model Complexes Elucidate the Role of the Proximal Hydrogen-Bonding Network in Cytochrome P450s.

Cytochrome (Cyt) P450s are an important class of enzymes with numerous functions in nature. The unique reactivity of these enzymes relates to their heme active sites with an axially bound, deprotonated cysteine (a "cysteinate") ligand (chemically speaking a thiolate). The heme-thiolate active sites further contain a number of conserved hydrogen-bonds (H-bonds) to the bound cysteinate ligand, which have been proposed to tune and stabilize the Fe-S bond.

Electron Paramagnetic Resonance Spectroscopy as a Probe of Hydrogen Bonding in Heme-Thiolate Proteins.

Despite utilizing a common cofactor binding motif, hemoproteins bearing a cysteine-derived thiolate ligand (heme-thiolate proteins) are involved in a diverse array of biological processes ranging from drug metabolism to transcriptional regulation. Though the origin of heme-thiolate functional divergence is not well understood, growing evidence suggests that the hydrogen bonding (H-bonding) environment surrounding the Fe-coordinating thiolate influences protein function. Outside of X-ray crystallography, few methods exist to characterize these critical H-bonding interactions.

Site-directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) "locked-off" state of the CO-sensing transcription factor CooA.

The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector-driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA.

GATA/Heme Multi-omics Reveals a Trace Metal-Dependent Cellular Differentiation Mechanism.

By functioning as an enzyme cofactor, hemoglobin component, and gene regulator, heme is vital for life. One mode of heme-regulated transcription involves amplifying the activity of GATA-1, a key determinant of erythrocyte differentiation. To discover biological consequences of the metal cofactor-transcription factor mechanism, we merged GATA-1/heme-regulated sectors of the proteome and transcriptome.

CO and NO bind to Fe(II) DiGeorge critical region 8 heme but do not restore primary microRNA processing activity.

The RNA-binding heme protein DiGeorge critical region 8 (DGCR8) and its ribonuclease partner Drosha cleave primary transcripts of microRNA (pri-miRNA) as part of the canonical microRNA (miRNA) processing pathway. Previous studies show that bis-cysteine thiolate-coordinated Fe(III) DGCR8 supports pri-miRNA processing activity, while Fe(II) DGCR8 does not. In this study, we further characterized Fe(II) DGCR8 and tested whether CO or NO might bind and restore pri-miRNA processing activity to the reduced protein.

Met(104) is the CO-replaceable ligand at Fe(II) heme in the CO-sensing transcription factor BxRcoM-1.

Both Met(104) and Met(105) are involved, either directly or indirectly, in the redox mediated ligand switch of the heme-dependent transcription factor, RcoM-1. Recent studies of Burkholderia xenovorans RcoM identified Cys(94) as the thiolate ligand in the Fe(III) state of the heme cofactor. Upon reduction, a neutral donor replaces Cys(94) trans to His(74).

Mechanism governing heme synthesis reveals a GATA factor/heme circuit that controls differentiation.

Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2).

Comparative study of enzyme activity and heme reactivity in Drosophila melanogaster and Homo sapiens cystathionine β-synthases.

Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5'-phosphate (PLP) for catalysis, and S-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear.

Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans.

The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2--C94S, C127S, and C130S--and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif.

Effect of the disease-causing R266K mutation on the heme and PLP environments of human cystathionine β-synthase.

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme.

Ethylene sensing by silver(I) salt-impregnated luminescent films.

Luminescent oligomers and polymers doped with silver(I) salts were used as optical sensors for ethylene and other gaseous small molecules. Films of poly(vinylphenylketone) (PVPK) or 1,4-bis(methylstyryl)benzene (BMSB) impregnated with AgBF(4), AgSbF(6), or AgB(C(6)F(5))(4) respond to ethylene exposures with a reversible emission quenching that is proportional to the pressure of the gas. Experiments with various analytes revealed that only gases capable of forming coordinate bonds with Ag(I) ions (i.

Ferric, not ferrous, heme activates RNA-binding protein DGCR8 for primary microRNA processing.

The RNA-binding protein DiGeorge Critical Region 8 (DGCR8) and its partner nuclease Drosha are essential for processing of microRNA (miRNA) primary transcripts (pri-miRNAs) in animals. Previous work showed that DGCR8 forms a highly stable and active complex with ferric [Fe(III)] heme using two endogenous cysteines as axial ligands. Here we report that reduction of the heme iron to the ferrous [Fe(II)] state in DGCR8 abolishes the pri-miRNA processing activity.

Cobalt cystathionine β-synthase: a cobalt-substituted heme protein with a unique thiolate ligation motif.

Human cystathionine β-synthase (hCBS), a key enzyme in the trans-sulfuration pathway, catalyzes the condensation of serine with homocysteine to produce cystathionine. CBS from higher organisms is the only known protein that binds pyridoxal-5'-phosphate (PLP) and heme. Intriguingly, the function of the heme in hCBS has yet to be elucidated.

DiGeorge critical region 8 (DGCR8) is a double-cysteine-ligated heme protein.

All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8.

Purification and characterization of cystathionine β-synthase bearing a cobalt protoporphyrin.

Human cystathionine β-synthase (CBS), a pivotal enzyme in the metabolism of homocysteine, is a pyridoxal-5'-phosphate-dependent enzyme that also contains heme, a second cofactor whose function is still unclear. One strategy for elucidation of heme function is its replacement with different metalloporphyrins or with porphyrins containing different substituent groups. This paper describes a novel expression approach and purification of cobalt CBS (CoCBS), which results in a high yield of fully active, high purity enzyme, in which heme is substituted by Co-protoporphyrin IX (CoPPIX).

Nitric oxide photogeneration from trans-Cr(cyclam)(ONO)(2)(+) in a reducing environment. activation of soluble guanylyl cyclase and arterial vasorelaxation.

The chromium(III) nitrito complex trans-Cr(cyclam)(ONO)(2)(+) (1) is a very promising photochemical precursor for nitric oxide delivery to physiological targets. Here, we demonstrate that visible wavelength excitation of 1 in solutions containing thiol reductants such as the biological antioxidant glutathione (GSH) leads to permanent reaction even under anaerobic conditions, resulting in high quantum yield NO release. The nitric oxide formed under such conditions is sufficient, even at muM concentrations of 1 and using a low-intensity light source, to activate the enzyme soluble guanylyl cyclase (sGC).

Guanidine hydrochloride-induced unfolding of the three heme coordination states of the CO-sensing transcription factor, CooA.

CooA is a heme-dependent CO-sensing transcription factor that has three observable heme coordination states. There is some evidence that each CooA heme state has a distinct protein conformation; the goal of this study was to characterize these conformations by measuring their structural stabilities through guanidine hydrochloride (GuHCl) denaturation. By studying the denaturation processes of the Fe(III) state of WT CooA and several variants, we were able to characterize independent unfolding processes for each domain of CooA.