Cross-linking Domain Research Articles

Bovine prolactin-related protein-I is anchored to the extracellular matrix through interactions with type IV collagen

The bovine placenta produces an array of proteins structurally similar to pituitary prolactin (PRL). At least ten genes of the bovine placental PRL family, including bovine placental lactogen (bPL) and ten bovine PRL-related protein-I to -X (bPRP-I to -X), encode hormones/cytokines predicted to be involved in the establishment and maintenance of pregnancy. Targets and biological roles for most members of the bovine PRL family have yet to be specified. This study focused on three members of bovine PRL family, bPL, bPRP-I, and bPRP-VI. An alkaline phosphatase (AP) tagging strategy was used to monitor interactions of the ligands with their targets. AP-bPRP-I and AP-bPRP-VI specifically bound to tissue sections of the bovine placentome. AP-bPRP-I and AP-bPRP-VI binding within the placentome mimicked the distribution of the extracellular matrix (ECM). Consequently, AP fusion protein binding to individual ECM components (heparin, laminin, fibronectin, type I collagen, and type IV collagen) was evaluated. AP-bPRP-I specifically bound to type IV collagen, but not to the other ECM components. AP-bPRP-VI exhibited weak interactions with ECM components, while AP-bPL and AP did not show significant binding to any of the ECM components. Binding of AP-bPRP-I to type IV collagen was concentration-dependent, influenced by salt concentrations, and specific to the N-terminal cross-linking domain (7S) of type IV collagen but not its triple-helical domain. The interaction of bPRP-I with type IV collagen suggests that bPRP-I accumulates in the ECM where it likely acts on cells traversing the bovine placentome.

Development and characterisation of novel cross-linked bio-elastomeric materials

Recombinantly-engineered elastin-like polypeptides (ELPs) possess many of the favourable attributes of the native elastin protein, making them an attractive option for designing biomaterials for tissue-engineering applications. The focus of this study was to synthesize and characterise the bulk material properties of two ELP sequences, ELP2 and ELP4, cross-linked with lysine diisocyanate (LDI). The two distinct ELPs consist of repeating hydrophobic and hydrophilic cross-linking domains in a block co-polymer structure, however, differ by the number of respective domains. Depending on the conditions sets for the cross-linking reactions, two different ELP-based materials were synthesized: a gel-like relatively non-porous material and a porous foam-like material, from both ELP sequences. The physical material properties were characterised by scanning electron microscopy, compression testing, differential calorimetry analysis and swelling analysis. The bulk material properties were found to vary depending on the ELP sequence investigated. ELP gels were also found to have a more dense solidified morphology, lower compressive moduli, higher melting temperature and greater swelling capacity than the porous ELP foams. These novel cross-linked bio-elastomeric materials show promising properties for soft tissue replacement, particularly in load-bearing applications.

Characterization of the Enzymatic Activity of the Actin Cross-linking Domain from the Vibrio cholerae MARTXVc Toxin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins, which alter host cells through a number of mechanisms resulting in diarrheal disease. Among the secreted toxins is the multifunctional, autoprocessing RTX toxin (MARTX(Vc)), which disrupts actin cytoskeleton by covalently cross-linking actin monomers into oligomers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we demonstrate unambiguously that ACD utilizes G- and not F-actin as a substrate for the cross-linking reaction and hydrolyzes one molecule of ATP per cross-linking event. Furthermore, major actin-binding proteins that regulate actin cytoskeleton in vivo do not block the cross-linking reaction in vitro. Cofilin inhibits the cross-linking of G- and F-actin, at a high mole ratio to actin but accelerates F-actin cross-linking at low mole ratios. DNase I completely blocks the cross-linking of actin, likely due to steric hindrance with one of the cross-linking sites on actin. In the context of the holotoxin, the inhibition of Rho by the Rho-inactivating domain of MARTX(Vc) (Sheahan, K. L., and Satchell, K. J. F. (2007) Cell. Microbiol. 9, 1324-1335) would accelerate F-actin depolymerization and provide G-actin, alone or in complex with actin-binding proteins, for cross-linking by ACD, ultimately leading to the observed rapid cell rounding.

Cyanogen Bromide Peptides of the Fibrillar Collagens I, III, and V and Their Mass Spectrometric Characterization: Detection of Linear Peptides, Peptide Glycosylation, and Cross-Linking Peptides Involved in Formation of Homo- and Heterotypic Fibrils

The network of the fibrillar collagens I, III, and V, extracted from fetal calf skin and cleaved with cyanogen bromide, was studied by means of ultraviolet matrix-assisted laser desorption ionization time-of-flight mass spectrometry (UV-MALDI MS). Nearly all of the expected cyanogen bromide peptides of the different alpha chains were detected. Distinct peptides are identified that can serve as a reference signal for the individual alpha-chains. Homo- and heterotypic cross-linking patterns, some of which have not been described before for bovine collagen, are indicated by comparison of the mass spectrometric data with documented amino acid sequences. Potential cross-linking mechanisms are discussed. For example, the mass spectrometric data suggest that the formation of heterotypic I/III and I/V fibrils is substantially determined by the telo-regions of type I collagen, which are covalently connected to the corresponding helical and nonhelical cross-linking domains of adjacent molecules either by 4D or 0D-stagger bonds. The chemical nature of the cross-links can be concluded. The data also indicate a disturbed formation of heterotypic fibrils. Finally, collagen glycosylation can also be identified.

Structure of the elastin gene.

The isolation and characterization of cDNAs encompassing the full length of chicken, cow, rat and human elastin mRNA have led to the elucidation of the primary structure of the respective tropoelastins. Large segments of the sequence are conserved but there are also considerable variations which range in extent from relatively small alterations, such as conservative amino acid substitutions, to variation in the length of hydrophobic segments and largescale deletions and insertions. In general, smaller differences are found among mammalian tropoelastins and greater ones between chicken and mammalian tropoelastins. Although only a single elastin gene is found per haploid genome, the primary transcript is subject to considerable alternative splicing, resulting in multiple tropoelastin isoforms. Functionally distinct hydrophobic and cross-link domains of the protein are encoded in separate exons which alternate in the gene. The introns of the human gene are rich in Alu repetitive sequences, which may be the site of recombinational events, and there are also several dinucleotide repeats, which may exhibit polymorphism and, therefore, be effective genetic markers. The 5' flanking region is G+C rich and contains potential binding sites for numerous modulating factors, but no TATA box or functional CAAT box. The basic promoter is contained within a 136 bp segment and transcription is initiated at multiple sites. These findings suggest that the regulation of elastin gene expression is complex and takes place at several levels.

Self-assembled elastin-like polypeptide particles

In this work, the self-assembly of a recombinant elastin-based block copolymer containing both hydrophobic and cross-linking domains from the human elastin protein was investigated. The particle formation and dynamic behavior were characterized using inverted microscopy and dynamic light scattering. The morphology and stability were evaluated using scanning and transmission electron microscopy. Above a critical temperature the molecules self-assembled into a bimodal distribution of nano- and micron-sized particles. The larger particles increased in size through coalescence. Micron-sized particle formation appeared largely reversible, although a self-assembly/disassembly hysteresis was observed. At high polyethylene glycol (PEG) concentrations particle coalescence and settling were reduced, particle stability seemed enhanced and PEG coated the particles. Particle stabilization was also achieved through covalent cross-linking using glutaraldehyde. This study laid the foundation for optimization of particle size and stability through modification of the solvent system and has shown that this family of elastin-based polypeptides holds potential for use as particulate drug carriers.

Bouncing Back From Elastin Deficiency

Large arteries are comprised of vascular smooth muscle cells (SMCs) embedded within a complex, cell-derived extracellular matrix. Collagen and elastic fibers, the major constituents of the vascular matrix, are secreted and assembled by SMCs and confer tensile and elastic properties. In the medial layer of elastic arteries, elastin forms concentric fenestrated lamellar layers that intercalate with alternating rings of SMCs to form functional lamellar units. 1 In the aorta, elastic fibers represent the largest component of the extracellular matrix, contributing up to 50% of aortic dry weight. 2 A series of elegant reports has demonstrated a critical role for elastin in the regulation of vascular morphogenesis in mice. Elastin (eln)-null mice die shortly after birth because of aortic obstruction by SMC proliferation. 3 Heterozygous mice (eln / ) are viable but produce 50% less elastin mRNA and protein these animals are hypertensive, exhibit thinner elastic lamellae, more lamellar units, decreased aortic compliance, and mild cardiac hypertrophy compared with eln / mice. 4 Extensive experimental studies have revealed that elevated arterial pressure is an adaptation to maintain cardiac output and tissue perfusion in spite of vessel stiffness,5 whereas the increase in lamellar unit number is an adaptation to normalize wall stress.6 In this issue of Circulation Research, Hirano et al report the phenotypic rescue of elastin-deficient mice by generation of a humanized elastin mouse. 7 Using a bacterial artificial chromosome encoding the entire human elastin gene (hBAC), they engineered mice to express functional human elastin (ELN) under the control of its native promoter. Several transgenic founder lines demonstrated at least 1 functional ELN insert and were capable of producing human elastin mRNA. Spatial and temporal expression of the human ELN transgene was similar to endogenous mouse elastin. Moreover, the hBAC mRNA product was appropriately spliced, and the protein was correctly secreted, assembled, and incorporated into mouse elastic fibers. At first glance, it may seem surprising that human elastin can substitute for mouse elastin, considering the differences in exon splicing and the lower than average amino acid conservation between species. In retrospect, however, Hirano et al might have expected the 2 proteins to be interchangeable, because it is primarily the structure of the elastin protein that is important for function. The alternating hydrophobic and crosslinking domains of elastin are conserved throughout vertebrate evolution, 8 and it is this repetitive domain structure that promotes the self-assembly of elastin into fibrillar structures,9 provides elastomeric properties,10,11 and stabilizes elastin to withstand repeated cycles of extension and recoil. 12 Transgenic expression of human elastin prevented lethality in the eln-null mouse, although the rescued animals expressed only 30% of normal elastin levels. In accordance with the lower elastin content, these animals exhibited a more severe cardiovascular phenotype than eln / mice, evidenced by stiffer vessels, higher blood pressure, and greater cardiac hypertrophy. Introduction of the hBAC into the eln / mice increased elastin content by 40%, and this resulted in a decrease in lamellar unit number and arterial pressure to levels measured in eln / mice, and partially restored vascular compliance. Taken together, these findings suggest a direct relationship between the amount of elastin produced (mouse and human combined) and the severity of the cardiovascular defect in mice. Thus, these mice might provide an elegant system for teasing apart the different thresholds for elastin expression which lead to specific abnormalities in elastic tissues. Indeed, these authors have also used this model to investigate elastin-dosing effects on lung development and susceptibility to smoke-induced emphysema.13

Population differences in elastin maturation in optic nerve head tissue and astrocytes.

Glaucomatous optic neuropathy is characterized by remodeling of the extracellular matrix with disorganization of elastic fibers in the optic nerve head (ONH). There are significant differences in prevalence of glaucomatous optic neuropathy between African Americans (AAs) and Caucasian Americans (CAs). The goal of this study was to evaluate differences in elastin synthesis and maturation in ONH tissue and cells of AA and CA donors with no eye disease, to provide a basis for underlying racial differences in susceptibility to elevated intraocular pressure. The amount of mature elastin in ONHs from each group of donors was evaluated by desmosine radioimmunoassay. The distribution of elastic fibers in ONH tissue was investigated by immunofluorescent staining. Elastin and lysyl oxidase mRNA levels and alternative splicing of elastin in ONH astrocytes were investigated by quantitative PCR. Tropoelastin protein expression was assessed by immunoblot analysis. ONHs from AA donors had significantly reduced levels of desmosine compared with those of CAs. In contrast, elastin mRNA and tropoelastin synthesis were elevated in ONH astrocytes from AA individuals. The inclusion of exon 23 in elastin mRNA and lysyl oxidase-like 2 mRNA levels was significantly reduced in astrocytes from AA compared with CA donors. A reduced number of cross-linking domains in elastin and decreased lysyl oxidase-like 2 expression leads to decreased amount of mature elastin in ONHs from healthy AA individuals compared with CA donors. These results suggest ELN and LOXL2 as candidate susceptibility genes for population-specific genetic risk of primary open-angle glaucoma (POAG).

RTX Toxin Actin Cross-Linking Activity in Clinical and Environmental Isolates of Vibrio cholerae

Vibrio cholerae strains from diverse O-antigen groups were evaluated for RTX toxin actin cross-linking activity. This study demonstrates that the actin cross-linking domain sequence is present within rtxA in the majority of clinical and environmental isolates tested, and the RTX toxin produced by these strains catalyzes the covalent cross-linking of cellular actin.

Unassociated Molecular Chains in Physically Crosslinked Gellan Gels

It was demonstrated that gellan chains unassociated with the crosslinking domains in the gels are released from gellan gels into the external solution at 10 °C. The concentration of released gellan chains in the external solution was estimated using a phenol-sulfuric acid method. The release of gellan chains was suppressed with increasing concentration of potassium chloride or calcium chloride in the external solution whilst it was enhanced with increasing concentration of tetramethylammonium chloride in the external solution. It was suggested from differential scanning calorimetry that the released gellan chains adopt a double helix conformation at 10 °C. The experimental results indicated that the double helical gellan chains, which can be released from the gels, are unassociated with the crosslinking domains composed of the lateral association of double helices in gellan gels. In conclusion, it was shown that the amount of unassociated gellan chains contained in the physical gel can be determined by measuring the concentration of released gellan chains in the external solution.

Inactivation of small Rho GTPases by the multifunctional RTX toxin from Vibrio cholerae

Many bacterial toxins target small Rho GTPases in order to manipulate the actin cytoskeleton. The depolymerization of the actin cytoskeleton by the Vibrio cholerae RTX toxin was previously identified to be due to the unique mechanism of covalent actin cross-linking. However, identification and subsequent deletion of the actin cross-linking domain within the RTX toxin revealed that this toxin has an additional cell rounding activity. In this study, we identified that the multifunctional RTX toxin also disrupts the actin cytoskeleton by causing the inactivation of small Rho GTPases, Rho, Rac and Cdc42. Inactivation of Rho by RTX was reversible in the presence of cycloheximide and by treatment of cells with CNF1 to constitutively activate Rho. These data suggest that RTX targets Rho GTPase regulation rather than affecting Rho GTPase directly. A novel 548-amino-acid region of RTX was identified to be responsible for the toxin-induced inactivation of the Rho GTPases. This domain did not carry GAP or phosphatase activities. Overall, these data show that the RTX toxin reversibly inactivates Rho GTPases by a mechanism distinct from other Rho-modifying bacterial toxins.

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

Domains 17–27 of tropoelastin contain key regions of contact for coacervation and contain an unusual turn-containing crosslinking domain

The central region of tropoelastin including domains 19–25 of human tropoelastin forms a hot-spot for contacts during the inter-molecular association of tropoelastin by coacervation [Wise, S.G., Mithieux, S.M., Raftery, M.J. and Weiss, A.S (2005). “Specificity in the coacervation of tropoelastin: solvent exposed lysines.” Journal of Structural Biology 149: 273-81.]. We explored the physical properties of this central region using a sub-fragment bordered by domains 17–27 of human tropoelastin (SHEL 17–27) and identified the intra- and inter-molecular contacts it forms during coacervation. A homobifunctional amine reactive crosslinker (with a maximum reach of 11 Å, corresponding to ∼ 7 residues in an extended polypeptide chain) was used to capture these contacts and crosslinked regions were identified after protease cleavage and mass spectrometry (MS) with MS/MS verification. An intermolecular crosslink formed between the lysines at positions 353 of each strand of tropoelastin at the lowest of crosslinker concentrations and was observed in all samples tested, suggesting that this residue forms an important initial contact during coacervation. At higher crosslinker concentrations, residues K425 and K437 showed the highest levels of involvement in crosslinks. An intramolecular crosslink between these K425 and K437, separated by 11 residues, indicated that a structural bend must serve to bring these residues into close proximity. These studies were complemented by small angle X-ray scattering studies that confirmed a bend in this important subfragment of the tropoelastin molecule.

The Actin Cross-linking Domain of the Vibrio cholerae RTX Toxin Directly Catalyzes the Covalent Cross-linking of Actin

Vibrio cholerae is a Gram-negative bacterial pathogen that exports enterotoxins to alter host cells and to elicit diarrheal disease. Among the secreted toxins is the multifunctional RTX toxin, which causes cell rounding and actin depolymerization by covalently cross-linking actin monomers into dimers, trimers, and higher multimers. The region of the toxin responsible for cross-linking activity is the actin cross-linking domain (ACD). In this study, we further investigated the role of the ACD in the actin cross-linking reaction. We show that the RTX toxin cross-links actin independently of tissue transglutaminase, thus eliminating an indirect model of ACD activity. We demonstrate that a fusion protein of the ACD and the N-terminal portion of lethal factor from Bacillus anthracis (LF(N)ACD) has cross-linking activity in vivo and in crude cell extracts. Furthermore, we determined that LF(N)ACD directly catalyzes the formation of covalent linkages between actin molecules in vitro and that Mg(2+) and ATP are essential cofactors for the cross-linking reaction. In addition, G-actin is proposed as a cytoskeletal substrate of the RTX toxin in vivo. Future studies of the in vitro cross-linking reaction will facilitate characterization of the enzymatic properties of the ACD and contribute to our knowledge of the novel mechanism of covalent actin cross-linking.

A Novel Dimer Interface and Conformational Changes Revealed by an X-ray Structure of B. subtilis SecA

The SecA ATPase moves polypeptides post-translationally across the plasma membrane of eubacteria, but the mechanism of transport is still unclear. We describe the crystal structure of a novel dimeric form of Bacillus subtilis SecA. Dimerization of SecA occurs at the prominent groove formed by the nucleotide binding domain 2 (nbd2) and the preprotein cross-linking (ppx) domain. The dimer interface is very large, burying approximately 5400 Å 2 of solvent accessible surface per monomer. Single cysteine disulfide cross-linking shows the presence of this novel SecA dimer in solution. In addition, other dimers also exist in solution, arguing that they all are in equilibrium with monomeric SecA and supporting the idea that the monomer may be the functional species. Dimerization of SecA causes an α-helix of one subunit to convert to a short β-strand that participates in β-sheet formation with strands in the other subunit. This conversion of secondary structure elements occurs close to the connection between the nbd1 and ppx domains, a potential site of interaction with translocation substrate. Comparing the different X-ray structures of B. subtilis SecA suggests that small changes in the nucleotide binding domains could be amplified via helix 1 of the helical scaffold domain (hsd) to generate larger movements of the domains involved in polypeptide binding.

Cooperativity between the Hydrophobic and Cross-linking Domains of Elastin

The principal protein component of the elastic fiber found in elastic tissues is elastin, an amorphous, cross-linked biopolymer that is assembled from a high molecular weight monomer. The hydrophobic and cross-linking domains of elastin have been considered separate and independent, such that changes to one region are not thought to affect the other. However, results from these solid-state 13C NMR experiments demonstrate that cooperativity in protein folding exists between the two domain types. The sequence of the EP20-24-24 polypeptide has three hydrophobic sequences from exons 20 and 24 of the soluble monomer tropoelastin, interspersed with cross-linking domains constructed from exons 21 and 23. In the middle of each cross-linking domain is a "hinge" sequence. When this pentapeptide is replaced with alanines, as in EP20-24-24[23U], its properties are changed. In addition to the expected increase in alpha-helical content and the resulting increase in rigidity of the cross-linking domains, changes to the organization of the hydrophobic regions are also observed. Using one-dimensional CPMAS (cross-polarization with magic angle spinning) techniques, including spectral editing and relaxation measurements, evidence for a change in dynamics to both domain types is observed. Furthermore, it is likely that the methyl groups of the leucines of the hydrophobic domains are also affected by the substitution to the hinge region of the cross-linking sequences. This cooperativity between the two domain types brings new questions to the phenomenon of coacervation in elastin polypeptides and strongly suggests that functional models for the protein must include a role for the cross-linking regions.

Correction of Pulmonary Abnormalities in Sftpd-/- Mice Requires the Collagenous Domain of Surfactant Protein D

Surfactant protein D (SP-D) is a member of the collectin family of innate defense proteins. Members of this family share four distinct structural domains: an N-terminal cross-linking domain, a collagenous domain, a neck region, and a carbohydrate recognition domain. In this study, the function of the collagenous domain was evaluated by expressing a SP-D collagen deletion mutant protein (rSftpdCDM) in wild type and SP-D null mice (Sftpd(-/-)). rSftpdCDM formed disulfide-linked trimers that further oligomerized into higher order structures. The mutant protein effectively bound carbohydrate and aggregated bacteria in vitro. Whereas rSftpdCDM did not disrupt pulmonary morphology or surfactant phospholipid levels in wild type mice, the mutant protein failed to rescue the emphysema or enlarged foamy macrophages that are characteristic of Sftpd(-/-) mice. Moreover, rSftpdCDM partitioned with small aggregate surfactant in a manner similar to SP-D, but rSftpdCDM did not correct the abnormal surfactant ultrastructure or phospholipid levels observed in Sftpd(-/-) mice. In contrast, rSftpdCDM completely corrected viral clearance and the abnormal inflammatory response that occurs following pulmonary influenza A challenge in Sftpd(-/-) mice. Our findings indicate that the collagen domain of SP-D is not required for assembly of disulfide-stabilized oligomers or the innate immune response to viral pathogens. The collagen domain of SP-D is required for the regulation of pulmonary macrophage activation, airspace remodeling, and surfactant lipid homeostasis.

Localizing α-Helices in Human Tropoelastin: Assembly of the Elastin “Puzzle”

Polyalanine cross-linking domains encoded by exons 6, 15, 17, 19, 21, 23, 25, 27, 29, 31 of human tropoelastin were synthesized, and their conformations were studied in different solutions and at different temperatures by CD and (1)H NMR. The results demonstrated the presence of poly-proline II helix (PPII) in aqueous solvent and of alpha-helical conformation in TFE. The (1)H NMR results allowed the precise localization of the helices along the peptide sequence. These data were further refined by prediction algorithms in order to take into account the reduced helix stability at the end of the peptides. Furthermore, the influence of flanking residues was checked by synthesizing and by determining the structure of a peptide spanning exon 31 coded domain and the first five residues of the following exon 32 coded domain. These studies, together with those previously published [Tamburro, A. M., Bochicchio, B., and Pepe, A. (2003) Biochemistry 42, 13147-62], are used to propose a coherent recomposition of the elastin pieces (domains) in order to give an acceptable solution to the elastin structure-function problem.

Structure of the p53 Core Domain Dimer Bound to DNA

The p53 tumor suppressor protein binds to DNA as a dimer of dimers to regulate transcription of genes that mediate responses to cellular stress. We have prepared a cross-linked trapped p53 core domain dimer bound to decamer DNA and have determined its structure by x-ray crystallography to 2.3A resolution. The p53 core domain subunits bind nearly symmetrically to opposite faces of the DNA in a head-to-head fashion with a loophelix motif making sequence-specific DNA contacts and bending the DNA by about 20 degrees at the site of protein dimerization. Protein subunit interactions occur over the central DNA minor groove and involve residues from a zinc-binding region. Analysis of tumor derived p53 mutations reveals that the dimerization interface represents a third hot spot for mutation that also includes residues associated with DNA contact and protein stability. Residues associated with p53 dimer formation on DNA are poorly conserved in the p63 and p73 paralogs, possibly contributing to their functional differences. We have used the dimeric protein-DNA complex to model a dimer of p53 dimers bound to icosamer DNA that is consistent with solution bending data and suggests that p53 core domain dimer-dimer contacts are less frequently mutated in human cancer than intra-dimer contacts.

Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

Cytochrome bd is a heterodimeric terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. For understanding the unique catalytic mechanism of the quinol oxidation, mass spectrometry was used to identify amino acid residue(s) that can be labeled with a reduced form of 2-azido-3-methoxy-5-methyl-6-geranyl-1,4-benzoquinone or 2-methoxy-3-azido-5-methyl-6-geranyl-1,4-benzoquinone. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry demonstrated that the photo inactivation of ubiquinol-1 oxidase activity was accompanied by the labeling of subunit I with both azidoquinols. The cross-linked domain was identified by reverse-phase high performance liquid chromatography of subunit I peptides produced by in-gel double digestion with lysyl endopeptidase and endoproteinase Asp-N. Electrospray ionization quadrupole time-of-flight mass spectrometry determined the amino acid sequence of the peptide (m/z 1047.5) to be Glu(278)-Lys(283), where a photoproduct of azido-Q(2) was linked to the carboxylic side chain of I-Glu(280). This study demonstrated directly that the N-terminal region of periplasmic loop VI/VII (Q-loop) is a part of the quinol oxidation site and indicates that the 2- and 3-methoxy groups of the quinone ring are in the close vicinity of I-Glu(280).