FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells.

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical 'chromatophore' vesicles. These bacterial 'organelles' are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc1 complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c2. Proximity between cytochrome bc1 and RC-LH1-PufX minimises quinone/quinol/cytochrome c2 diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc1 with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc1 complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc1 observed by spectroscopy, and explain how quinols/quinones and cytochrome c2 shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.

A microbubble-sparged yeast propagation-fermentation process for bioethanol production.

BACKGROUND: Industrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation-fermentation bioethanol process was developed and analysed. RESULTS: Aeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations. CONCLUSIONS: The performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation.

Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli.

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans.IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid.

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc1 complex (cytbc1) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc1 complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc1 complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc1 to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc1 complexes retain their native dimeric structure and co-purify with 56±6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b6f complex (cytb6f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc1/cytb6f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q0 and Qi sites.

Micrometre and nanometre scale patterning of binary polymer brushes, supported lipid bilayers and proteins.

Binary polymer brush patterns were fabricated via photodeprotection of an aminosilane with a photo-cleavable nitrophenyl protecting group. UV exposure of the silane film through a mask yields micrometre-scale amine-terminated regions that can be derivatised to incorporate a bromine initiator to facilitate polymer brush growth via atom transfer radical polymerisation (ATRP). Atomic force microscopy (AFM) and imaging secondary ion mass spectrometry (SIMS) confirm that relatively thick brushes can be grown with high spatial confinement. Nanometre-scale patterns were formed by using a Lloyd's mirror interferometer to expose the nitrophenyl-protected aminosilane film. In exposed regions, protein-resistant poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMEMA) brushes were grown by ATRP and used to define channels as narrow as 141 nm into which proteins could be adsorbed. The contrast in the pattern can be inverted by (i) a simple blocking reaction after UV exposure, (ii) a second deprotection step to expose previously intact protecting groups, and (iii) subsequent brush growth via surface ATRP. Alternatively, two-component brush patterns can be formed. Exposure of a nitrophenyl-protected aminosilane layer either through a mask or to an interferogram, enables growth of an initial POEGMEMA brush. Subsequent UV exposure of the previously intact regions allows attachment of ATRP initiator sites and growth of a second poly(cysteine methacrylate) (PCysMA) brush within photolithographically-defined micrometre or nanometre scale regions. POEGMEMA brushes resist deposition of liposomes, but fluorescence recovery after photobleaching (FRAP) studies confirm that liposomes readily rupture on PCysMA "corrals" defined within POEGMEMA "walls". This leads to the formation of highly mobile supported lipid bilayers that exhibit similar diffusion coefficients to lipid bilayers formed on surfaces such as glass.

From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups.

We show that sequential protein deposition is possible by photodeprotection of films formed from a tetraethylene-glycol functionalized nitrophenylethoxycarbonyl-protected aminopropyltriethoxysilane (NPEOC-APTES). Exposure to near-UV irradiation removes the protein-resistant protecting group, and allows protein adsorption onto the resulting aminated surface. The protein resistance was tested using proteins with fluorescent labels and microspectroscopy of two-component structures formed by micro- and nanopatterning and deposition of yellow and green fluorescent proteins (YFP/GFP). Nonspecific adsorption onto regions where the protecting group remained intact was negligible. Multiple component patterns were also formed by near-field methods. Because reading and writing can be decoupled in a near-field microscope, it is possible to carry out sequential patterning steps at a single location involving different proteins. Up to four different proteins were formed into geometric patterns using near-field lithography. Interferometric lithography facilitates the organization of proteins over square cm areas. Two-component patterns consisting of 150 nm streptavidin dots formed within an orthogonal grid of bars of GFP at a period of ca. 500 nm could just be resolved by fluorescence microscopy.

Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy.

The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip-sample interaction in both lateral and vertical directions. Here, long-range tip-sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic "organelles", chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles.

Strong Coupling of Localized Surface Plasmons to Excitons in Light-Harvesting Complexes.

Gold nanostructure arrays exhibit surface plasmon resonances that split after attaching light harvesting complexes 1 and 2 (LH1 and LH2) from purple bacteria. The splitting is attributed to strong coupling between the localized surface plasmon resonances and excitons in the light-harvesting complexes. Wild-type and mutant LH1 and LH2 from Rhodobacter sphaeroides containing different carotenoids yield different splitting energies, demonstrating that the coupling mechanism is sensitive to the electronic states in the light harvesting complexes. Plasmon-exciton coupling models reveal different coupling strengths depending on the molecular organization and the protein coverage, consistent with strong coupling. Strong coupling was also observed for self-assembling polypeptide maquettes that contain only chlorins. However, it is not observed for monolayers of bacteriochlorophyll, indicating that strong plasmon-exciton coupling is sensitive to the specific presentation of the pigment molecules.

Versatile thiol-based reactions for micrometer- and nanometer-scale photopatterning of polymers and biomolecules.

Thiol-based chemistry provides a mild and versatile tool for surface functionalization. In the present work, mercaptosilane films were patterned by utilizing UV-induced photo-oxidation of the thiol to yield sulfonate groups via contact and interferometric lithography (IL). These photo-generated sulfonic acid groups were used for selective immobilization of amino-functionalized molecules after activation with triphenylphosphine ditriflate (TPPDF). Moreover, protein-resistant poly(oligoethyleneglycolmethacrylate) (POEGMA) brushes were grown from the intact thiol groups by a surface-induced polymerization reaction. Exploiting both reactions it is possible to couple amino-labelled nitrilotriacetic acid (NH2-NTA) to sulfonate-functionalized regions, enabling the site-specific binding of green fluorescent protein (GFP) to regions defined lithographically, while exploiting the protein-resistant character of POEGMA brushes to prevent non-specific protein adsorption to previously masked areas. The outstanding reactivity of thiol groups paves the way towards novel strategies for the fabrication of complex protein nanopatterns beyond thiol-ene chemistry.

Fast, simple, combinatorial routes to the fabrication of reusable, plasmonically active gold nanostructures by interferometric lithography of self-assembled monolayers.

We describe a fast, simple method for the fabrication of reusable, robust gold nanostructures over macroscopic (cm(2)) areas. A wide range of nanostructure morphologies is accessible in a combinatorial fashion. Self-assembled monolayers of alkylthiolates on chromium-primed polycrystalline gold films are patterned using a Lloyd's mirror interferometer and etched using mercaptoethylamine in ethanol in a rapid process that does not require access to clean-room facilities. The use of a Cr adhesion layer facilitates the cleaning of specimens by immersion in piranha solution, enabling their repeated reuse without significant change in their absorbance spectra over two years. A library of 200 different nanostructures was prepared and found to exhibit a range of optical behavior. Annealing yielded structures with a uniformly high degree of crystallinity that exhibited strong plasmon bands. Using a combinatorial approach, correlations were established between the preannealing morphologies (determined by the fabrication conditions) and the postannealing optical properties that enabled specimens to be prepared "to order" with a selected localized surface plasmon resonance. The refractive index sensitivity of gold nanostructures formed in this way was found to correlate closely with measurements reported for structures fabricated by other methods. Strong enhancements were observed in the Raman spectra of tetra-tert-butyl-substituted phthalocyanine. The shift in the position of the plasmon band after site-specific attachment of histidine-tagged green fluorescent protein (His-GFP) and bacteriochlorophyll a was measured for a range of nanostructured films, enabling the rapid identification of the one that yielded the largest shift. This approach offers a simple route to the production of durable, reusable, macroscopic arrays of gold nanostructures with precisely controllable morphologies.

Zwitterionic poly(amino acid methacrylate) brushes.

A new cysteine-based methacrylic monomer (CysMA) was conveniently synthesized via selective thia-Michael addition of a commercially available methacrylate-acrylate precursor in aqueous solution without recourse to protecting group chemistry. Poly(cysteine methacrylate) (PCysMA) brushes were grown from the surface of silicon wafers by atom-transfer radical polymerization. Brush thicknesses of ca. 27 nm were achieved within 270 min at 20 °C. Each CysMA residue comprises a primary amine and a carboxylic acid. Surface zeta potential and atomic force microscopy (AFM) studies of the pH-responsive PCysMA brushes confirm that they are highly extended either below pH 2 or above pH 9.5, since they possess either cationic or anionic character, respectively. At intermediate pH, PCysMA brushes are zwitterionic. At physiological pH, they exhibit excellent resistance to biofouling and negligible cytotoxicity. PCysMA brushes undergo photodegradation: AFM topographical imaging indicates significant mass loss from the brush layer, while XPS studies confirm that exposure to UV radiation produces surface aldehyde sites that can be subsequently derivatized with amines. UV exposure using a photomask yielded sharp, well-defined micropatterned PCysMA brushes functionalized with aldehyde groups that enable conjugation to green fluorescent protein (GFP). Nanopatterned PCysMA brushes were obtained using interference lithography, and confocal microscopy again confirmed the selective conjugation of GFP. Finally, PCysMA undergoes complex base-catalyzed degradation in alkaline solution, leading to the elimination of several small molecules. However, good long-term chemical stability was observed when PCysMA brushes were immersed in aqueous solution at physiological pH.

Bactericidal activity of the human skin fatty acid cis-6-hexadecanoic acid on Staphylococcus aureus.

Human skin fatty acids are a potent aspect of our innate defenses, giving surface protection against potentially invasive organisms. They provide an important parameter in determining the ecology of the skin microflora, and alterations can lead to increased colonization by pathogens such as Staphylococcus aureus. Harnessing skin fatty acids may also give a new avenue of exploration in the generation of control measures against drug-resistant organisms. Despite their importance, the mechanism(s) whereby skin fatty acids kill bacteria has remained largely elusive. Here, we describe an analysis of the bactericidal effects of the major human skin fatty acid cis-6-hexadecenoic acid (C6H) on the human commensal and pathogen S. aureus. Several C6H concentration-dependent mechanisms were found. At high concentrations, C6H swiftly kills cells associated with a general loss of membrane integrity. However, C6H still kills at lower concentrations, acting through disruption of the proton motive force, an increase in membrane fluidity, and its effects on electron transfer. The design of analogues with altered bactericidal effects has begun to determine the structural constraints on activity and paves the way for the rational design of new antistaphylococcal agents.

Integration of energy and electron transfer processes in the photosynthetic membrane of Rhodobacter sphaeroides.

Photosynthesis converts absorbed solar energy to a protonmotive force, which drives ATP synthesis. The membrane network of chlorophyll-protein complexes responsible for light absorption, photochemistry and quinol (QH2) production has been mapped in the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides using atomic force microscopy (AFM), but the membrane location of the cytochrome bc1 (cytbc1) complexes that oxidise QH2 to quinone (Q) to generate a protonmotive force is unknown. We labelled cytbc1 complexes with gold nanobeads, each attached by a Histidine10 (His10)-tag to the C-terminus of cytc1. Electron microscopy (EM) of negatively stained chromatophore vesicles showed that the majority of the cytbc1 complexes occur as dimers in the membrane. The cytbc1 complexes appeared to be adjacent to reaction centre light-harvesting 1-PufX (RC-LH1-PufX) complexes, consistent with AFM topographs of a gold-labelled membrane. His-tagged cytbc1 complexes were retrieved from chromatophores partially solubilised by detergent; RC-LH1-PufX complexes tended to co-purify with cytbc1 whereas LH2 complexes became detached, consistent with clusters of cytbc1 complexes close to RC-LH1-PufX arrays, but not with a fixed, stoichiometric cytbc1-RC-LH1-PufX supercomplex. This information was combined with a quantitative mass spectrometry (MS) analysis of the RC, cytbc1, ATP synthase, cytaa3 and cytcbb3 membrane protein complexes, to construct an atomic-level model of a chromatophore vesicle comprising 67 LH2 complexes, 11 LH1-RC-PufX dimers & 2 RC-LH1-PufX monomers, 4 cytbc1 dimers and 2 ATP synthases. Simulation of the interconnected energy, electron and proton transfer processes showed a half-maximal ATP turnover rate for a light intensity equivalent to only 1% of bright sunlight. Thus, the photosystem architecture of the chromatophore is optimised for growth at low light intensities.

EfeUOB (YcdNOB) is a tripartite, acid-induced and CpxAR-regulated, low-pH Fe2+ transporter that is cryptic in Escherichia coli K-12 but functional in E. coli O157:H7.

Escherichia coli possesses iron transporters specific for either Fe2+ or Fe3+. Although Fe2+ is far more soluble than Fe3+, it rapidly oxidizes aerobically at pH > or = 7. Thus, FeoAB, the major Fe2+ transporter of E. coli, operates anaerobically. However, Fe2+ remains stable aerobically under acidic conditions, although a low-pH Fe2+ importer has not been previously identified. Here we show that ycdNOB (efeUOB) specifies the first such transporter. efeUOB is repressed at high pH by CpxAR, and is Fe2+-Fur repressed. EfeU is homologous to the high-affinity iron permease, Ftr1p, of Saccharomyces cerevisiae and other fungi. EfeO is periplasmic with a cupredoxin N-terminal domain; EfeB is also periplasmic and is haem peroxidase-like. All three Efe proteins are required for Efe function. The efeU gene of E. coli K-12 is cryptic due to a frameshift mutation - repair of the single-base-pair deletion generates a functional EfeUOB system. In contrast, the efeUOB operon of the enterohaemorrhagic strain, O157:H7, lacks any frameshift and is functional. A 'wild-type' K-12 strain bearing a functional EfeUOB displays a major growth advantage under aerobic, low-pH, low-iron conditions when a competing metal is provided. 55Fe transport assays confirm the ferrous iron specificity of EfeUOB.

Preliminary X-ray diffraction analysis of YcdB from Escherichia coli: a novel haem-containing and Tat-secreted periplasmic protein with a potential role in iron transport.

YcdB is a periplasmic haem-containing protein from Escherichia coli that has a potential role in iron transport. It is currently the only reported haem-containing Tat-secreted substrate. Here, the overexpression, purification, crystallization and structure determination at 2.0 A resolution are reported for the apo form of the protein. The apo-YcdB structure resembles those of members of the haem-dependent peroxidase family and thus confirms that YcdB is also a member of this family. Haem-soaking experiments with preformed apo-YcdB crystals have been optimized to successfully generate haem-containing YcdB crystals that diffract to 2.9 A. Completion of model building and structure refinement are under way.

Probing the unusual oxidation/reduction behavior of Paracoccus pantotrophus cytochrome cd1 nitrite reductase by replacing a switchable methionine heme iron ligand with histidine.

A M106H variant, where M106 is a c-type heme iron axial ligand, of cytochrome cd(1) nitrite reductase is an inactive protein in vivo. Expression of the holoprotein in Paracoccus pantotrophus required generation of nitric oxide during cell growth through simultaneous expression of an exogenous copper nitrite reductase from Alcaligenes faecalis. In the absence of the latter protein, only a semi-apo form of M106H cytochrome cd(1) was formed. Thus it was demonstrated that expression of the chromosomal nir genes for d(1) heme biosynthesis in P. pantotrophus is NO-dependent, probably mediated by the transcription factor NNR, and a route to low or zero activity mutants had been established. The value of such variants for mechanistic studies on cytochrome cd(1) is illustrated by the use of M106H to demonstrate that the d(1) heme potential can be resolved and measured at approximately +175 mV with the c heme shifted to -60 mV, consistent with its bishistidinyl coordination. The unusual highly cooperative and strongly hysteretic redox titration of the wild type is lost in the M106H protein. The same c heme midpoint potential was observed in a M106H variant of a c-domain construct. The difference between d(1) heme and c heme redox potentials has allowed preparation of a M106H protein with oxidized c heme and reduced d(1) heme. This one electron reduced form will reduce nitrite to nitric oxide, but the latter remains bound to the resulting fully oxidized enzyme.

Feo--transport of ferrous iron into bacteria.

Bacteria commonly utilise a unique type of transporter, called Feo, to specifically acquire the ferrous (Fe2+) form of iron from their environment. Enterobacterial Feo systems are composed of three proteins: FeoA, a small, soluble SH3-domain protein probably located in the cytosol; FeoB, a large protein with a cytosolic N-terminal G-protein domain and a C-terminal integral inner-membrane domain containing two 'Gate' motifs which likely functions as the Fe2+ permease; and FeoC, a small protein apparently functioning as an [Fe-S]-dependent transcriptional repressor. We provide a review of the current literature combined with a bioinformatic assessment of bacterial Feo systems showing how they exhibit common features, as well as differences in organisation and composition which probably reflect variations in mechanisms employed and function.

Paracoccus pantotrophus NapC can reductively activate cytochrome cd1 nitrite reductase.

The oxidized "as isolated" form of Paracoccus pantotrophus cytochrome cd1 nitrite reductase has a bis-histidinyl coordinated c heme and a histidine/tyrosine coordinated d1 heme. This form of the enzyme has previously been shown to be kinetically incompetent. Upon reduction, the coordination of both hemes changes and the enzyme is kinetically activated. Here, we show that P. pantotrophus NapC, a tetraheme c-type cytochrome belonging to a large family of such proteins, is capable of reducing, and hence activating, "as isolated" cytochrome cd1. NapC is the first protein from P. pantotrophus identified as being capable of this activation step and, given the periplasmic co-location and co-expression of the two proteins, is a strong candidate to be a physiological activation partner.

Identification of two domains and distal histidine ligands to the four haems in the bacterial c-type cytochrome NapC; the prototype connector between quinol/quinone and periplasmic oxido-reductases.

NapC is a tetra-haem member of a family of bacterial membrane-anchored multi-haem c -type cytochromes implicated in electron transfer between membrane quinols and periplasmic enzymes. The water-soluble tetra-haem fragment of Paracoccus pantotrophus NapC has been expressed as a periplasmic protein (NapC(sol)) in Paracoccus denitrificans, P. pantotrophus and Escherichia coli. Site-specific mutagenesis of NapC(sol), combined with spectroscopic studies, suggests that each haem iron centre has bis -histidinyl co-ordination. Four proximal ligands arise from each of four Cys-Xaa-Xaa-Cys-His haem-binding motifs; candidates for the four distal ligands are His(81), His(99), His(174) and His(194). NapC(H81A), NapC(H99A), NapC(H174A) and NapC(H194A) mutants (with alanine substituted for each of the four candidate residues) have all been purified from E. coli. In each case, one of the haems has become high-spin, as judged by the presence of a broad absorption band between 620 nm and 650 nm for the oxidized cytochrome; this feature is absent for wild-type protein and presumably arises because of the absence of the distal histidine ligand from one of the haems. NapC(H81A) and NapC(H174A) are less well expressed in E. coli than NapC(H99A) and NapC(H194A) and cannot be detected when expressed in P. denitrificans or P. pantotrophus. In vitro and in vivo complementation studies demonstrate that the soluble periplasmic NapC can mediate electron transfer from quinols to the periplasmic nitrate reductase. This capacity was retained in vitro with the NapC(H99A) and NapC(H194A) mutants but was lost in vivo. A model for the structural organization of NapC(sol) into two domains, each containing a di-haem pair, is proposed. In this model, each haem pair obtains one distal haem ligand from its own domain and a second from the other domain. The suggestion of two domains is supported by observations that the 24 kDa NapC(sol) cleaves to yield a 12 kDa haem-staining band. Determination of the cleavage site showed it was between two equally sized di-haem domains predicted from sequence analysis.