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Well-resolved, Soret band excited resonance Raman spectra were measured from the fully oxidized and fully reduced cytochrome c oxidase from beef heart and Paracoccus denitrificans. The vibrational patterns in the marker band region (1450-1700 cm-1) were analyzed, and a complete assignment of heme a and heme a3 vibrational modes is presented, permitting a detailed structural comparison of the mammalian and bacterial enzymes. Similar frequencies of the porphyrin modes for the reduced heme a and the reduced and oxidized heme a3 are found, indicating a close relationship of the ground-state conformations in all oxidase species studied. In oxidized heme a, however, significant frequency differences are observed and interpreted in terms of a ruffled porphyrin structure in the three- and two-subunit forms of the Paracoccus enzyme compared to the planar heme a of beef heart oxidase. The structural distortions, which also perturb the conformation of the formyl substituent and its electronic coupling with the porphyrin, reflect the specific heme-protein interactions at heme a. Since in the fully reduced state heme a appears to be largely planar in all oxidase species, the redox-linked conformational transition requires a more drastic rearrangement of the heme a-protein interactions in the bacterial than in the mammalian oxidase. For both heme a and heme a3 in the reduced state and for heme a3 in the oxidize state, frequency, intensity, and bandwidth differences of the formyl stretching vibration and intensity differences of some porphyrin modes are noted between the three oxidase forms. The same modes are also affected by quaternary structure changes in the bovine oxidase caused by different detergents and isolation procedures. These effects are attributed to differences of the dielectric properties of the heme environment, due to subtle structural changes in the heme pockets, induced by protein-protein interactions of subunit III with subunits I and/or II.

Implantable medical devices such as catheters are indispensable in the management of critically and chronically ill patients for the administration of electrolytes, drugs, parenteral nutrients, blood components or drainage of secretions and pus. Artificial heart valves, prosthetics, ceramics, metals and bone cements are standard implants. All of these implants save human lives and enhance quality of life. At the same time they are the leading cause for millions of primary nosocomial bloodstream infections with substantial morbidity and mortality 1 . A property common to all these biomaterials is the ease by which they are colonized by pathogenic and nonpathogenic microorganisms, often requiring immediate removal.Several methods have been devised to decrease the risk of foreign body-associated infections. These include the use of meticulous hygienic precautions, the development of hydrophilic materials to minimize bacterial adhesion and impregnation with antiseptics and antibiotics. Silver, in particular free silver ions 2 , is well known for its powerful and broad-spectrum antimicrobial activity still allowing the independent use of therapeutic antibiotics. The investigation of the antimicrobial activity of implants containing silver as an antimicrobial agent is difficult because many silver compounds are poorly water soluble, resulting in low concentrations of silver ions released into the surrounding medium. Therefore, the antimicrobial efficacy of polymers impregnated with elementary silver cannot be tested by routine agar diffusion measurements 3 . Like other procedures 4,5 , the agar diffusion technique was also inappropriate for a simultaneous highthroughput screening of silver prototypes.Reliable in vitro methods for antimicrobial activity testing of surfaces are essential for the development of new anti-infective biomaterials. Cell proliferation is an important step in the course of infection 6 and must be included in any evaluation procedure. To date, assays 7-10 have focused on the monitoring of bacterial adherence but lack an analysis of the microbial proliferatory behavior. For a precise testing of antimicrobial efficacy three independent aspects must be considered: adhesion (the test must detect and quantify adherent microorganisms); proliferation (the test should assay the potential of adherent bacteria for proliferation); and detection of bactericidal and bacteriostatic activity.Here, we introduce a new technique for testing antimicrobial properties of biomaterials using a microplate system (Fig. 1). As a selected example, we show in vitro data for the antimicrobial activity of silver polymers, which correlate positively with multicenter clinical trials 11 . Implications of the approach.The number of new biomaterials in medicine is steadily growing. Highly efficient in vitro methods are required for quality control, screening and product improvement. Such comparative techniques should meet the following requirements: a quantification method to monitor microbial adherence; a sensitive and reproducib...

Cytochrome c oxidase from the bacterium Paracoccus denitrcjicans, while being related to the mitochondrial enzyme in many ways, consists of only two to three different subunits. For the identification of its genes, a Paracoccus DNA library was constructed and screened with specific antibodies for expression of cloned inserts in E. coli. A positive clone expressing immunoreactive products in the molecular mass region of authentic subunit I1 revealed a high homology of its DNA-deduced amino acid sequence with subunit I1 sequences of the mitochondrial oxidases; several typical features, such as the transmembrane folding pattern and the presumed copper-binding site, are highly conserved between prokaryotic and mitochondrial polypeptides. A comparison with peptide sequencing data of the purified subunit established the presence of a characteristic N-terminal extension as well as a longer C terminus in the initial translation product of the Paracoccus subunit; by mass spectroscopy, the first N-terminally blocked residue of the mature polypeptide was identified as a pyroglutamate. No code abnormalities, but a highly specific codon usage were observed; no evidence for a localization of the subunit I gene directly adjacent to this gene has been obtained.Cytochrome c oxidase is a key enzyme in the electrontransport chains of mitochondria and many bacteria. It catalyzes the transfer of electrons from the one-electron donor cytochrome c to the four-electron acceptor dioxygen; this reaction is coupled to the extrusion of protons from the mitochondrial matrix (or the cytoplasma in the case of bacteria) to generate an electrochemical gradient across the membrane (for recent reviews on the mitochondrial enzyme, see [l -61). Interest in the bacterial enzymes has been stimulated by a number of reports (reviewed in [7 -91) showing that the structure of the prokaryotic oxidases is far less complex than that of the mitochondrial enzymes which, depending on the source, are composed of from 6 (lower eukaryotes) to up to 13 subunits (mammalian mitochondria). Typically only two to three different subunits are seen in isolated bacterial heme aa3-type oxidases; such preparations show full activity in electron transport, and several of them are also active in proton translocation (see [9]). Since evidence for homology of subunits from mitochondrial and prokaryotic oxidases has been presented, these findings have greatly supported the notion that the three largest, mitochondrially coded subunits constitute the catalytic core also in the mitochondrial enzyme.The oxidase from the bacterium Paracoccus denitrificans [lo -121 is among the best-studied prokaryotic oxidases and may be viewed as a structurally simple model system for the mitochondrial enzyme: as isolated, the Paracoccus enzyme is composed of two different subunits of 45 kDa and 28-30 kDa, efficiently oxidizes reduced bacterial or mitochon- drial cytochrome c, and acts as a redox-coupled proton pump both in proteoliposomes and in whole cells [13 -151. Apart from considerable furthe...

We describe the construction and characterization of Essentially three features have made the gram-negative soil bacterium Paracoccus denitrificans an interesting organism for bioenergetic research and, in particular, a model organism for the mitochondrial electron transport. (i) Its close relationship to present-day mitochondria, indicated by a large number of mostly physiological and bioenergetic arguments, prompted John and Whatley (23,24) to assign this bacterium a hypothetical precursor role in endosymbiotic organelle development; this early assumption of a close evolutionary homology has recently been confirmed by more direct evidence on phylogenetic grounds (59). (ii) At least two respiratory complexes, cytochrome c reductase (see below) and cytochrome c oxidase (17,30,32) isolated from this bacterium in a functional state, have basic enzymatic properties almost indistinguishable from those of their mitochondrial counterparts, but are far less complex in their structure. (iii) Due to the availability of the genes for their subunits, these enzymes are amenable to site-specific mutagenesis experiments to study stucture-function relationships and thus increase our knowledge on basic electron transport and energy transduction mechanisms both in bacteria and in mitochondria.The cytochrome bc, complex (complex III; ubiquinol: cytochrome c oxidoreductase [EC 1.10.2.2]) is an obligate component in the electron transport chain of mitochondria and, in an analogous form, of photosynthetic organisms (19,20,43,58). Moreover, several bacterial species have recently been shown to contain a functionally homologous complex, notably Rhodobacter species (8,13,14) and Paracoccus denitrificans (4, 33, 60). Three different subunits carrying the redox centers of this integral membrane enzyme, cytochromes b and cl and the so-called Rieske protein with its iron-sulfur center (FeS), are common to all bc, complexes. Whereas the well-studied enzyme complexes from mitochondria typically contain a large number of additional polypeptides of uncertain function, the bacterial enzymes mentioned above are composed of only the three essential redox center subunits. However, at least for Para-* Corresponding author. coccus spp., full enzymatic competence of this structurally simple complex has been reported in a reconstituted system, for both its electron transport and energy transduction capacity (60, 61). Molecular details of these two coupled processes have not been elucidated in mitochondrial bc1 complexes, but hypotheses for structure-function relationships as well as tentative functional assignments for certain residues have been made (10,22,38,45,52,57). Such assumptions can be tested in bacteria by performing sitedirected mutagenesis on the isolated genes, followed by insertion into a suitable host to obtain expression of the redox complex. In Paracoccus spp., the genes for the bc1 subunits have been cloned and sequenced (27), and, as in Rhodobacter spp. (8, 13), have been shown to be organized in an operon structure, subsequent...

Paracoccus has approx. 15 subunits [28]. The transcriptional organization of this NDH-l-cluster is presently unknown. NQ01 and NQ02 code for flavoproteins, NQ03 to NQ06 are genes for iron-sulfur proteins whereas NQ07 to NQ014 code for polypeptides of the hydrophobic protein fraction [28]. The gene order in this cluster is NQ07, NQ06, NQ05, NQ04, NO02, NQ01 NQ03, NQ08 NQ09, NQ010, NQ011, NQO12, NQ013, and NQ014.Upstream of NQ07 an open reading frame with homology to Escherichia coli uvrA is found. A reading frame with homology to E. coli biotin acetyl-CoA carboxylase ligase has been found downstream of the cluster [28].Sequences for two genes, NQ01 [29], encoding the NADH-binding subunit, and NQ02 [30], which codes for an iron-sulfur protein, have been published. Two URFs have been found; sequence similarities of URF2 to ctaG of the Paracoccus cta-operon suggest a possible role as an assembly protein, most probably in the attachment of iron [29,30,55]. Because of its simpler structure the Paracoccus complex is likely to play an important part in investigating inhibitory agents or the mode of complex formation during assembly. Mutant strains are presently not available.

The purpose of this study was to establish a reliable and cost-effective microplate proliferation assay for in vitro antimicrobial testing of bone cement samples. Cement samples devoid of antimicrobial agents, loaded with 2% gentamicin or with different concentrations of high-porosity silver, were incubated in a 96-well microplate with several staphylococcal, Pseudomonas aeruginosa, and Enterococcus faecium isolates exhibiting different susceptibilities to gentamicin. After being rinsed, the samples were brought into a soy medium in which adherent cells on the cement surface either were killed by the antimicrobial surface or started to release clonal counterparts. The medium was monitored in real time by recording a time proliferation curve for each well. Microplate testing revealed no antibacterial effect of plain bone cement. The antibacterial activity of gentamicin-loaded bone cement was shown by the microplate test to depend on the gentamicin susceptibilities of the strains. The effect of high-porosity silver was dose dependent. Bactericidal activity against all tested strains was found for bone cement loaded with 1% high-porosity silver. The accuracy of this new proliferation assay was shown by the high correlation between the types of proliferation curves and antibiotic susceptibility. In contrast to routine agar diffusion testing, it assesses the dynamic response of microorganisms to antimicrobial agents in biomaterials and allows high-throughput screening and detection of antimicrobial properties of poorly water-soluble compounds like silver.Infections in total joint arthroplasty are devastating situations (19), and many strategies have been undertaken to reduce infection rates, including the use of helmet aspirator suits (22), laminar airflow (5, 10, 18), and prophylactic intravenous antibiotics (9, 13, 15). Loading polymethylmethacrylate (PMMA) bone cement with antibiotics to reduce infection rates has also been postulated in the literature (6,16,24,26).As in all other fields of medicine, new ideas in orthopedics should be evaluated stepwise, with in vitro and subsequent in vivo investigations of antimicrobial properties occurring before clinical trials. Therefore, testing the antimicrobial activity of bone cement with new antibiotics or other antimicrobial agents should begin with in vitro studies. Agar diffusion testing has been the standard method for many years for in vitro assessment of antibiotic-loaded bone cements (17,23,27,28). However, the assessment by agar diffusion testing of the anti-infective activities of some antimicrobial agents requires accurately defined standard conditions (20). For example, large molecules, such as vancomycin and teicoplanin, harbor a reduced diffusion capacity and their susceptibilities are difficult to determine exactly. Other anti-infective agents, e.g., fosfomycin, are able to interact with components of the culture medium in agar plates, resulting in reduced activity. Therefore, adequate culture media are required (20). New anti-infective agents like microdis...

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