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Within the framework of an investigation of the electronic structure of oxidized high-potential iron-sulfur proteins (HiPIP), we have studied the HiPIP II from Ectothiorhodospira vacuolata, which was known to have a peculiar temperature dependence of the 1H NMR isotropic hyperfine shifts. The signals of the cysteine ligand protons have been sequence specifically assigned through NOE, NOESY, and TOCSY experiments. Nine hyperfine-shifted signals are observed: seven in the downfield and two in the upfield region. They have been assigned to the eight beta-CH2 protons of the four coordinated cysteines and to one alpha-CH cysteine proton. The two most downfield-shifted signals belong to the beta-CH2 protons of Cys 63 (Chromatium vinosum numbering) and the two upfield protons to those of Cys 43. These two pairs of protons show a Curie-type temperature dependence of the hyperfine shifts. Among the remaining five downfield-shifted signals, three show a Curie-type temperature dependence and two have an anti-Curie temperature dependence. The former are assigned to the beta-CH2 and alpha-CH protons of Cys 77 and the latter to the beta-CH2 protons of Cys 46. The shift patterns are thus similar, in a sequence-specific sense, to those of the analogous proteins from C. vinosum and Rhodocyclus gelatinosus, whereas they differ from those of Rhodocyclus globiformis HiPIP and even more from those of Ectothiorhodospira halophila HiPIP II. Oxidized HiPIPs can be formally viewed as containing a cluster of four ferric ions plus one extra electron. We present here a model based on a chemical equilibrium, fast on the NMR time scale, between two species, both of which contain a pair of iron(III) ions and a mixed-valence pair but are differently oriented within the protein frame. The EPR data are also discussed in the light of the debate on the nature of the different species detected at low temperature. The interpretation of the whole set of data on HiPIPs in the light of the present model is compared with that based on previous models.

Nitrous oxide reductase (N 2 OR) is the terminal enzyme involved in denitrification by microbes. No threedimensional structural information has been published for this enzyme. We have isolated and characterised N 2 OR from Alcaligenes xylosoxidans (AxN 2 OR) as a homodimer of M r 134 000 containing seven to eight copper atoms per dimer. Comparison of sequence and compositional data with other N 2 ORs suggests that AxN 2 OR is typical and can be expected to have similar domain folding and subunit structure to other members of this family of enzymes. We present synchrotron X-ray-scattering data, analysed using a model-independent method for shape restoration, which gave a < 20 A Ê resolution structure of the enzyme in solution, providing a glimpse of the structure of any N 2 OR and shedding light on the molecular architecture of the molecule. The specific activity of AxN 2 OR was < 6 mmol of N 2 O reduced´min ±1´( mg of protein) ±1 ; N 2 OR activity showed both base and temperature activation. The visible spectrum exhibited an absorption maximum at 550 nm with a shoulder at 635 nm. On oxidation with K 3 Fe(CN) 6 , the absorption maximum shifted to 540 nm and a new shoulder at 480 nm appeared. Reduction under anaerobic conditions resulted in the formation of an inactive blue form of the enzyme with a broad absorption maximum at 650 nm. As isolated, the enzyme shows an almost featureless EPR spectrum, which changes on oxidation to give an almost completely resolved seven-line hyperfine signal in the g II region, g = 2.18, with A II = 40 G, consistent with the enzyme being partially reduced as isolated. Both the optical and EPR spectra of the oxidized enzyme are characteristic of the presence of a Cu A centre.

The effects of increasing concentrations of C1-, CIO;, and HCO; on the redox potential of Rhodopseudomonas palustris cytochrome c2 indicate that the two polyatomic anions bind specifically to the protein at one site, while chloride simply exerts an ionic atmosphere effect. The change in E" upon specific anion binding allows us to probe for the influence of surface charges on the redox potential of cytochromes c. The decrease in redox potential at null ionic strength (dEF=,,) due to anion neutralization of one positive surface charge was found to be 23 mV with perchlorate and 33 mV with bicarbonate. These values compare reasonably well with previous theoretical predictions and estimates of the effect of charge alteration on the E" values in cytochromes c chemically modified or mutated at surface lysines. These AE" values, determined on the unmodified protein, are unprecedented for c-type cytochromes. The anion-induced chemical shift changes of the hyperfine-shifted heme 'H-NMR resonances of the oxidized protein yield lower limit values of 53 M-' and 18 M for the affinity constant for specific HCO; and CIO; binding, respectively.Keywords: cytochromes c; electrochemistry; NMR; redox potential; protein dielectric.Cytochromes cz are electron transfer proteins involved in bacterial photosynthesis as electron donors to the reaction center [l-41. They are related to mitochondria1 cytochromes c by structure and sequence and, like their honiologues, are basic proteins. Therefore, the effect of anions on their physicochemical properties and on the kinetics of electron transfer to the biological partners is of considerable interest. A wealth of data has been collected for mitochondrial cytochromes c on the effect that the ionic composition of the medium exerts on the conformation of both redox states 15-91, on the kinetics of intermolecular electron transfer with biological partners [I 0-131, and on the redox potential and the thermodynamic parameters of the redox reaction [14-171. Anion interaction with this class of proteins has also been investigated with reference to the location of the sites of specific anion binding and to the determination of the affinity constants [lS, 17-20]. Less is known on the ionic strength dependence of the physicochemical properties of cytochromes c,. Most of the work was devoted to the study of the effect of the ionic composition of the medium on the kinetics of electron transfer with biological partners, in order to understand the determinants of complex information [21-241. We describe here the effects of CI-, HCO, and HCO; on the redox potential and proton NMR spectra of Rhodopseudomonas palustris cytochrome c2. The first two anions were chosen for their ubiquity in living systems, while perchlorate is of interest due to its chaotropic properties. We do not know of any other report on anion binding to this class of bacterial cytochromes. The results indicate that, at neutral pH, perchlorate and bicarbonate bind to the protein surface at one site, at variance with chloride which appears no...

The pH-induced protein conformational transitions and changes in the ligation state of the heme iron in cytochrome c, from Rhodopseudomonas palustris were monitored by electrochemical and spectroscopic measurements. In the pH range 1.5-11, the E" values (and/or the peak potentials) determined by cyclic voltammetry, the electronic spectra and the hyperfine-shifted 'H-NMR resonances of the protein are sensitive to a number of acidlbase equilibria. In particular, four equilibria have been determined for the oxidized protein with pK, values of 2.5, 5.5, 6.6 and 9.0. The lowest pK, most probably involves disruption of both axial heme iron bonds and protein unfolding. The subsequent pK, is associated with a low-pH oxidation of the protein by dioxygen, which is accompanied by a conformational change. The equilibrium with an apparent pK, of 6.6 modulates the E" values without determining any detectable spectral change and most likely involves the acidbase equilibrium of an histidine residue in close vicinity of the heme (possibly His53). Finally, the alkaline ionization is due to the replacement of the methionine axially bound to the heme iron with a stronger (most probably N-donor) ligand. The reduced alkaline form is unstable and spontaneously converts to the neutral reduced form with a kinetic constant of 0.98 s-' at pH 9.2.Keywords: cytochromes c ; electrochemistry ; redox potential ; NMR ; protein conformation. c, cytochromes (cyt c,) are class-I cytochromes involved in the electron-transport chains of photosynthesis and respiration in most photosynthetic non-sulfur purple bacteria and some nonphototropic bacteria [ 1 -41. They are structurally homologous to eukaryotic c cytochromes, although showing less than 40% sequence similarity [5, 61. c2 cytochromes are divided in two groups according to their molecular masses ; the 'mitochondrial' cyt c,, with a molecular mass of about 12 kDa, which resemble more closely the eukaryotic analogues, and the 'long' cyt c2 with a molecular mass around 13-14 kDa (71. The redox potentials of cyt c2 are, in general, 50-100 mV more positive than those of the eukaryotic proteins and show greater variability [8, 91. To date, the X-ray structures of cyt c2 isolated from Rhodospirillum rubrum, Rhodobacter capsulatus, Paracoccus denitrificans, Rhodobacter sphaeroides and Rhodopseudomonas viridis have been solved [lo-151. A single covalently bound heme lies near the N-terminus of the polypeptide chain, with a low-spin iron axially coordinated by a histidine residue adjacent to the heme attachment site, and a methionine residue near the C-terminus. Five a-helices, arranged around the heme in such a way as to leave one heme edge exposed to the solvent, determine the main folding motif. Differences in local structural features in the c2 family, particularly concerning the hydrogen-bonding network and solvent structure in the heme pocket, also in comparison with the eukaryotic analogues, are of use to investigate the factors controlling redox potentials and electron-transfer rates in Corresp...

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM. In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.

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