Pulse dipolar ESR of doubly labeled mini TAR DNA and its annealing to mini TAR RNA.

Pulse dipolar electron-spin resonance in the form of double electron electron resonance was applied to strategically placed, site-specifically attached pairs of nitroxide spin labels to monitor changes in the mini TAR DNA stem-loop structure brought on by the HIV-1 nucleocapsid protein NCp7. The biophysical structural evidence was at Ångstrom-level resolution under solution conditions not amenable to crystallography or NMR. In the absence of complementary TAR RNA, double labels located in both the upper and the lower stem of mini TAR DNA showed in the presence of NCp7 a broadened distance distribution between the points of attachment, and there was evidence for several conformers. Next, when equimolar amounts of mini TAR DNA and complementary mini TAR RNA were present, NCp7 enhanced the annealing of their stem-loop structures to form duplex DNA-RNA. When duplex TAR DNA-TAR RNA formed, double labels initially located 27.5 Å apart at the 3'- and 5'-termini of the 27-base mini TAR DNA relocated to opposite ends of a 27 bp RNA-DNA duplex with 76.5 Å between labels, a distance which was consistent with the distance between the two labels in a thermally annealed 27-bp TAR DNA-TAR RNA duplex. Different sets of double labels initially located 26-27 Å apart in the mini TAR DNA upper stem, appropriately altered their interlabel distance to ~35 Å when a 27 bp TAR DNA-TAR RNA duplex formed, where the formation was caused either through NCp7-induced annealing or by thermal annealing. In summary, clear structural evidence was obtained for the fraying and destabilization brought on by NCp7 in its biochemical function as an annealing agent and for the detailed structural change from stem-loop to duplex RNA-DNA when complementary RNA was present.

The internal dynamics of mini c TAR DNA probed by electron paramagnetic resonance of nitroxide spin-labels at the lower stem, the loop, and the bulge.

Electron paramagnetic resonance (EPR) at 236.6 and 9.5 GHz probed the tumbling of nitroxide spin probes in the lower stem, in the upper loop, and near the bulge of mini c TAR DNA. High-frequency 236.6 GHz EPR, not previously applied to spin-labeled oligonucleotides, was notably sensitive to fast, anisotropic, hindered local rotational motion of the spin probe, occurring approximately about the NO nitroxide axis. Labels attached to the 2'-aminocytidine sugar in the mini c TAR DNA showed such anisotropic motion, which was faster in the lower stem, a region previously thought to be partially melted. More flexible labels attached to phosphorothioates at the end of the lower stem tumbled isotropically in mini c TAR DNA, mini TAR RNA, and ψ(3) RNA, but at 5 °C, the motion became more anisotropic for the labeled RNAs, implying more order within the RNA lower stems. As observed by 9.5 GHz EPR, the slowing of nanosecond motions of large segments of the oligonucleotide was enhanced by increasing the ratio of the nucleocapsid protein NCp7 to mini c TAR DNA from 0 to 2. The slowing was most significant at labels in the loop and near the bulge. At a 4:1 ratio of NCp7 to mini c TAR DNA, all labels reported tumbling times of >5 ns, indicating a condensation of NCp7 and TAR DNA. At the 4:1 ratio, pulse dipolar EPR spectroscopy of bilabels attached near the 3' and 5' termini showed evidence of an NCp7-induced increase in the 3'-5' end-to-end distance distribution and a partially melted stem.

EPR-ENDOR characterization of (17O, 1H, 2H) water in manganese catalase and its relevance to the oxygen-evolving complex of photosystem II.

The synthesis of efficient water-oxidation catalysts demands insight into the only known, naturally occurring water-oxidation catalyst, the oxygen-evolving complex (OEC) of photosystem II (PSII). Understanding the water oxidation mechanism requires knowledge of where and when substrate water binds to the OEC. Mn catalase in its Mn(III)-Mn(IV) state is a protein model of the OEC's S(2) state. From (17)O-labeled water exchanged into the di-µ-oxo di-Mn(III,IV) coordination sphere of Mn catalase, CW Q-band ENDOR spectroscopy revealed two distinctly different (17)O signals incorporated in distinctly different time regimes. First, a signal appearing after 2 h of (17)O exchange was detected with a 13.0 MHz hyperfine coupling. From similarity in the time scale of isotope incorporation and in the (17)O µ-oxo hyperfine coupling of the di-µ-oxo di-Mn(III,IV) bipyridine model (Usov, O. M.; Grigoryants, V. M.; Tagore, R.; Brudvig, G. W.; Scholes, C. P. J. Am. Chem. Soc. 2007, 129, 11886-11887), this signal was assigned to µ-oxo oxygen. EPR line broadening was obvious from this (17)O µ-oxo species. Earlier exchange proceeded on the minute or faster time scale into a non-µ-oxo position, from which (17)O ENDOR showed a smaller 3.8 MHz hyperfine coupling and possible quadrupole splittings, indicating a terminal water of Mn(III). Exchangeable proton/deuteron hyperfine couplings, consistent with terminal water ligation to Mn(III), also appeared. Q-band CW ENDOR from the S(2) state of the OEC was obtained following multihour (17)O exchange, which showed a (17)O hyperfine signal with a 11 MHz hyperfine coupling, tentatively assigned as µ-oxo-(17)O by resemblance to the µ-oxo signals from Mn catalase and the di-µ-oxo di-Mn(III,IV) bipyridine model.

Thermodynamic equilibrium between blue and green copper sites and the role of the protein in controlling function.

A combination of spectroscopies and density functional theory calculations indicate that there are large temperature-dependent absorption spectral changes present in green nitrite reductases (NiRs) due to a thermodynamic equilibrium between a green and a blue type 1 (T1) copper site. The axial methionine (Met) ligand is unconstrained in the oxidized NiRs, which results in an enthalpically favored (DeltaH approximately 4.6 kcal/mol) Met-bound green copper site at low temperatures, and an entropically favored (TDeltaS approximately 4.5 kcal/mol, at room temperature) Met-elongated blue copper site at elevated temperatures. In contrast to the NiRs, the classic blue copper sites in plastocyanin and azurin show no temperature-dependent behavior, indicating that a single species is present at all temperatures. For these blue copper proteins, the polypeptide matrix opposes the gain in entropy that would be associated with the loss of the weak axial Met ligand at physiological temperatures by constraining its coordination to copper. The potential energy surfaces of Met binding indicate that it stabilizes the oxidized state more than the reduced state. This provides a mechanism to tune down the reduction potential of blue copper sites by >200 mV.

The role of arginine-127 at the proximal NO-binding site in determining the electronic structure and function of 5-coordinate NO-heme in cytochrome c' of Rhodobacter sphaeroides.

Cytochrome c' is a heme protein from a denitrifying variant of Rhodobacter sphaeroides which may serve to store and transport metabolic NO while protecting against NO toxicity. Its heme site bears resemblance through its 5-coordinate NO-binding capability to the regulatory site in soluble guanylate cyclase. A conserved arginine (Arg-127) abuts the 5-coordinate NO-heme binding site, and the alanine mutant R127A provided insight into the role of the Arg-127 in establishing the electronic structure of the heme-NO complex and in modifying the heme-centered redox potential and NO-binding affinity. By comparison to R127A, the wild-type Arg-127 was determined to increase the heme redox potential, diminish the NO-binding affinity, perturb and diminish the 14NO hyperfine coupling determined by ENDOR (electron nuclear double resonance), and increase the maximal electronic g-value. The larger isotropic NO hyperfine and the smaller maximal g-value of the R127A mutant together predicted that the Fe-N-O bond angle in the mutant is larger than that of the Arg-127-containing wild-type protein. Deuterium ENDOR provided evidence for exchangeable H/D consistent with hydrogen bonding of Arg-127, but not Ala-127, to the O of the NO. Proton ENDOR features previously assigned to Phe-14 on the distal side of the heme were unperturbed by the proximal side R127A mutation, implying the localized nature of that mutational perturbation at the proximal, NO-binding side of the heme. From this work two functions of positively charged Arg-127 emerged: the first was to maintain the KD of the cytochrome c' in the 1 microM range, and the second was to provide a redox potential that enhances the stability of the ferrous heme.

Anisotropic Fermi couplings due to large unquenched orbital angular momentum: Q-band (1)H, (14)N, and (11)B ENDOR of bis(trispyrazolylborate) cobalt(II).

We report Q-band ENDOR of (1)H, (14)N, and (11)B at the g( parallel) extreme of the EPR spectrum of bis(trispyrazolylborate) cobalt(II) [Co(Tp)(2)] and two structural analogs. This trigonally symmetric, high-spin (hs) S = 3/2 Co(II) complex shows large unquenched ground-state orbital angular momentum, which leads to highly anisotropic electronic g-values (g( parallel) = 8.48, g( perpendicular) = 1.02). The large g-anisotropy is shown to result in large dipolar couplings near g( parallel) and uniquely anisotropic (14)N Fermi couplings, which arise from spin transferred to the nitrogen 2s orbital (2.2%) via antibonding interactions with singly occupied metal d(x(2)-y(2)) and d(z(2)) orbitals. Large, well-resolved (1)H and (11)B dipolar couplings were also observed. Taken in concert with our previous X-band ENDOR measurements at g( perpendicular) ( Myers, W. K.; et al. Inorg. Chem. 2008, 47, 6701-6710 ), the present data allow a detailed analysis of the dipolar hyperfine tensors of two of the four symmetry distinct protons in the parent molecule. In the substituted analogs, changes in hyperfine coupling due to altered metal-proton distances give further evidence of an anisotropic Fermi contact interaction. For the pyrazolyl 3H proton, the data indicate a 0.2 MHz anisotropic contact interaction and approximately 4% transfer of spin away from Co(II). Dipolar coupling also dominates for the axial boron atoms, consistent with their distance from the Co(II) ion, and resolved (11)B quadrupolar coupling showed approximately 30% electronic inequivalence between the B-H and B-C sp(3) bonds. This is the first comprehensive ENDOR study of any hs Co(II) species and lays the foundation for future development.

Spectroscopic and computational studies of nitrite reductase: proton induced electron transfer and backbonding contributions to reactivity.

A combination of spectroscopy and DFT calculations has been used to define the geometric and electronic structure of the nitrite bound type 2 (T2) copper site at high and low pH in nitrite reductase from Rhodobacter sphaeroides. At high pH there is no electron transfer from reduced type 1 (T1) to the nitrite bound T2 copper, while protonation triggers T1 --> T2 electron transfer and generation of NO. The DFT calculated reaction coordinate for the N-O bond cleavage in nitrite reduction by the reduced T2 copper suggests that the process is best described as proton transfer triggering electron transfer. Bidentate nitrite binding to copper is calculated to play a major role in activating the reductive cleavage of the nitrite bond through backbonding combined with stabilization of the (-)OH product by coordination to the Cu(2+).

HIV-1 nucleocapsid protein NCp7 and its RNA stem loop 3 partner: rotational dynamics of spin-labeled RNA stem loop 3.

The tumbling dynamics of a 20-mer HIV-1 RNA stem loop 3 spin-labeled at the 5' position were probed in the nanosecond time range. This RNA interacted with the HIV-1 nucleocapsid Zn-finger protein, 1-55 NCp7, and specialized stopped-flow EPR revealed concomitant kinetics of probe immobilization from milliseconds to seconds. RNA stem loop 3 is highly conserved in HIV, while NCp7 is critical to HIV-RNA packaging and annealing. The 5' probe did not perturb RNA melting or the NCp7/RNA interaction monitored by gel shift and fluorescence. The 5'-labeled RNA tumbled with a subnanosecond isotropic correlation time (approximately 0.60 ns at room temperature) reflecting both local viscosity-independent bond rotation of the probe and viscosity-dependent diffusion of 40-60% of the RNA. The binding of NCp7 to spin-labeled RNA stem loop 3 in a 1:1 ratio increased the spin-labeled tumbling time by about 40%. At low ionic strength with a ratio of NCp7 to RNA >or=3 (i.e., an NCp7 to nucleotide ratio or=3:1 complex also required intact Zn fingers. Stopped-flow EPR kinetics with NCP7/RNA mixed at a 4:1 ratio showed the major phase of NCp7 interaction with RNA stem loop 3 occurred within 4 ms, a second phase occurred with a time constant of approximately 30 ms, and a slower immobilization, possibly concomitant with large complex formation, proceeded over seconds. This work points the way for spin-labeling to investigate oligonucleotide-protein complexes, notably those lacking precise stoichiometry, that are requisite for viral packaging and genome fabrication.

Exchangeable proton ENDOR as a probe of the redox-active iron center in activated bleomycin and ferric bleomycin.

Activated bleomycin (ABLM) is a drug-Fe(iii)-hydroperoxide complex kinetically competent in DNA attack (via H4' abstraction). This intermediate is relatively stable, but its spontaneous conversion to ferric bleomycin (Fe(iii)·BLM) is poorly characterized because no observable intermediate product accumulates. The Fe(iii)·BLM formed cryophotolytically from ABLM and kept at 77 K was remarkably similar by EPR and ENDOR criteria to Fe(iii)·BLM formed from Fe(iii) + BLM solution. The notable ENDOR criteria were the ENDOR frequencies and features of orientation-selected, strongly hyperfine-coupled, exchangeable protons associated with the environs of the iron within <3.5 Å of paramagnetic Fe(iii) in Fe(iii)·BLM and ABLM. Cryophotolytic conversion of activated bleomycin to its ferric bleomycin product in the frozen solid is a sign that the reaction requires only constrained local proton rearrangements. We have characterized the metal-proton distances and orientations of the protons in that rearrangement, especially noting that these protons are of mechanistic importance in the ambient temperature conversion of ABLM to Fe(iii)·BLM in concert with a directed radical-forming attack on DNA.

Cryogenic photolysis of activated bleomycin to ferric bleomycin.

Activated bleomycin (ABLM) is a drug--Fe(III)-hydroperoxide complex kinetically competent in DNA attack (via H4' abstraction). This intermediate is relatively stable, but its spontaneous conversion to ferric bleomycin (Fe(III).BLM) is poorly characterized because no observable intermediate product accumulates. Light was shown to trigger ABLM attack on DNA in liquid at -30 degrees C, so ABLM was irradiated (at its 350 nm ligand-to-metal charge-transfer transition) at 77 K to stabilize possible intermediates. ABLM photolysis (quantum yield, Phi = 0.005) generates two kinds of product: Fe(III).BLM (with no detectable intermediate) and one or more minor (1-2%) radical O-Fe-BLM byproduct, photostable at 77 K. Adding DNA, even without its target H4', increases the quantum yield of ABLM conversion >10-fold while suppressing the observed radical yield. Since cryogenic solid-phase reactions can entail only constrained local rearrangement, the reaction(s) converting ABLM to Fe(III).BLM must be similarly constrained.

Porphyrin-phospholipid liposomes permeabilized by near-infrared light.

The delivery of therapeutic compounds to target tissues is a central challenge in treating disease. Externally controlled drug release systems hold potential to selectively enhance localized delivery. Here we describe liposomes doped with porphyrin-phospholipid that are permeabilized directly by near-infrared light. Molecular dynamics simulations identified a novel light-absorbing monomer esterified from clinically approved components predicted and experimentally demonstrated to give rise to a more stable porphyrin bilayer. Light-induced membrane permeabilization is enabled with liposomal inclusion of 10 molar % porphyrin-phospholipid and occurs in the absence of bulk or nanoscale heating. Liposomes reseal following laser exposure and permeability is modulated by varying porphyrin-phospholipid doping, irradiation intensity or irradiation duration. Porphyrin-phospholipid liposomes demonstrate spatial control of release of entrapped gentamicin and temporal control of release of entrapped fluorophores following intratumoral injection. Following systemic administration, laser irradiation enhances deposition of actively loaded doxorubicin in mouse xenografts, enabling an effective single-treatment antitumour therapy.

Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): a study of doubly-spin-labeled T4 lysozyme.

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution. We compared the results from rapidly freeze-quenched (≤100 µs) samples to those from commonly shock-frozen (slow freeze, 1 s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

Rotational dynamics of HIV-1 nucleocapsid protein NCp7 as probed by a spin label attached by peptide synthesis.

2,2,6,6-Tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label was attached at the N-terminal position to interrogate the dynamics of the HIV-1 nucleocapsid Zn-finger protein, NCp7. NCp7 is a 6.4-kDa 55-mer critical to the recognition, packaging, and efficient reverse transcription of viral RNA that has stem-loop structures, such as the RNA stem-loop 3 used in this work. The NCp7, made by solid-phase peptide synthesis with TOAC incorporated into the alpha-carbon backbone at the N-terminal "0" position, showed analytical purity and biological activity. Electron Paramagnetic Resonance (EPR) spectra of the N-terminal TOAC indicated rapid temperature-sensitive motion of the probe (< or =0.33 ns correlation time) on the flexible N-terminal segment. This N-terminal TOAC-NCp7 reported a RNA-NCp7 interaction at a 1:1 ratio of NCp7 to RNA, which caused the tumbling time to be slowed from about 0.3 ns to about 0.5 ns. NCp7 is a largely disordered protein that adapts to its RNA targets. However, as shown by circular dichroism, > or =90% trifluoroethanol [(TFE), an alpha-helix enhancer] caused the TOAC-NCp7 without zinc in its fingers to change to a fully helical conformation, while the TOAC spin label was concurrently reporting a tumbling time of well over a nanosecond, as the N-terminal TOAC became inflexibly enfolded. Even with TFE present, the existence of intact Zn-finger regions in NCp7 prevented complete formation of helical structure, as shown by circular dichroism, and decreased the N-terminal TOAC tumbling time, as shown by EPR. This study demonstrated TOAC at the N-terminal of NCp7 to be a reporter for the considerable conformational lability of NCp7. (

EPR-ENDOR of the Cu(I)NO complex of nitrite reductase.

With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. (14)N- and (15)N-nitrite substrates gave rise to characteristic (14)NO and (15)NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with (63)Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated approximately 50% spin on the N of NO and approximately 17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cdelta1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cdelta1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 A from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).

ENDOR of NO-ligated cytochrome c'.

The five-coordinate NO-bound heme in cytochrome c' from an overexpressing variant of denitrifying R. sphaeroides 2.4.3 was investigated by proton, nitrogen, and deuterium Q-band ENDOR (electron nuclear double resonance). ENDOR was a direct probe of the unpaired electron density on the nitrogen of NO and, as measured across the EPR line shape, showed a hyperfine coupling range from 36 to 44 MHz for 14NO and 51 to 63 MHz for 15NO. The smallest NO coupling occurred at an electronic g-tensor axis perpendicular to the FeNO plane, and the largest hyperfine coupling occurred in the FeNO plane where the highest nitrogen valence spin density is located. The isotropic component of the NO hyperfine coupling indicated that the electron spin on the NO is not simply in a pi orbital having only 2p character but is in an orbital having 2s and 2p character in a 1:2 ratio. ENDOR frequencies from heme meso-protons, assigned with reference to porphyrin models, were determined to result from an anisotropic hyperfine tensor. This tensor indicated the orientation of the heme with respect to the FeNO plane and showed that the FeNO plane bisects the heme N-Fe-N 90 degrees angle. ENDOR provided additional structural information through dipolar couplings, as follows: (1) to the nearest proton of the Phe14 ring, approximately 3.1 A away from the heme iron, where Phe14 is positioned to occlude binding of NO as a 6th (distal) ligand; (2) to exchangeable deuterons assigned to Arg127 which may H-bond with the proximal NO ligand.

ENDOR investigation of the liganding environment of mixed-spin ferric cytochrome c'.

The electronic structure of the 5-coordinate quantum-mechanically mixed-spin (sextet-quartet) heme center in cytochrome c' was investigated by electron nuclear double resonance (ENDOR), a technique not previously applied to this mixed-spin system. Cytochrome c' was obtained from overexpressing variants of Rhodobacter sphaeroides 2.4.3. ENDOR for this study was done at the g(//) = 2.00 extremum where single-crystal-like, well-resolved spectra prevail. The heme meso protons of cytochrome c' showed a contact interaction that implied spin delocalization arising from the heme (d(z)(2)) orbital enhanced by iron out-of-planarity. An exchangeable proton ENDOR feature appeared from the proximal His123 Ndelta hydrogen. This Ndelta hydrogen, which crystallographically has no hydrogen-bonding partner and thus belongs to a neutral imidazole, showed a larger hyperfine coupling than the corresponding hydrogen-bonded Ndelta proton from metmyoglobin. The unique residue Phe14 occludes binding of a sixth ligand in cytochrome c', and ENDOR from a proton of the functionally important Phe14 ring, approximately 3.3 A away from the heme iron, was detected. ENDOR of the nitrogen ligand hyperfine structure is a direct probe into the sigma-antibonding (d(z)(2)) and (d(x)(2)-d(y)(2)) orbitals whose energies alter the relative stability and admixture of sextet and quartet states and whose electronic details were thus elucidated. ENDOR frequencies showed for cytochrome c' larger hyperfine couplings to the histidine nitrogen and smaller hyperfine couplings to the heme nitrogens than for high-spin ferric hemes. Both of these findings followed from the mixed-spin ground state, which has less (d(x)(2)-d(y)(2)) character than have fully high-spin ferric heme systems.

Regulation and function of cytochrome c' in Rhodobacter sphaeroides 2.4.3.

Cytochrome c' (Cyt c') is a c-type cytochrome with a pentacoordinate heme iron. The gene encoding this protein in Rhodobacter sphaeroides 2.4.3, designated cycP, was isolated and sequenced. Northern blot analysis and beta-galactosidase assays demonstrated that cycP transcription increased as oxygen levels decreased and was not repressed under denitrifying conditions as observed in another Rhodobacter species. CO difference spectra performed with extracts of cells grown under different conditions revealed that Cyt c' levels were highest during photosynthetic denitrifying growth conditions. The increase in Cyt c' under this condition was higher than would be predicted from transcriptional studies. Electron paramagnetic resonance analysis of whole cells demonstrated that Cyt c' binds NO during denitrification. Mass spectrometric analysis of nitrogen oxides produced by cells and purified protein did not indicate that Cyt c' has NO reductase activity. Taken together, these results suggest a model where Cyt c' in R. sphaeroides 2.4.3 may shuttle NO to the membrane, where it can be reduced.

Role of the coordinating histidine in altering the mixed valency of Cu(A): an electron nuclear double resonance-electron paramagnetic resonance investigation.

The binuclear Cu(A) site engineered into Pseudomonas aeruginosa azurin has provided a Cu(A)-azurin with a well-defined crystal structure and a CuSSCu core having two equatorial histidine ligands, His120 and His46. The mutations His120Asn and His120Gly were made at the equatorial His120 ligand to understand the histidine-related modulation to Cu(A), notably to the valence delocalization over the CuSSCu core. For these His120 mutants Q-band electron nuclear double resonance (ENDOR) and multifrequency electron paramagnetic resonance (EPR) (X, C, and S-band), all carried out under comparable cryogenic conditions, have provided markedly different electronic measures of the mutation-induced change. Q-band ENDOR of cysteine C(beta) protons, of weakly dipolar-coupled protons, and of the remaining His46 nitrogen ligand provided hyperfine couplings that were like those of other binuclear mixed-valence Cu(A) systems and were essentially unperturbed by the mutation at His120. The ENDOR findings imply that the Cu(A) core electronic structure remains unchanged by the His120 mutation. On the other hand, multifrequency EPR indicated that the H120N and H120G mutations had changed the EPR hyperfine signature from a 7-line to a 4-line pattern, consistent with trapped-valence, Type 1 mononuclear copper. The multifrequency EPR data imply that the electron spin had become localized on one copper by the His120 mutation. To reconcile the EPR and ENDOR findings for the His120 mutants requires that either: if valence localization to one copper has occurred, the spin density on the cysteine sulfurs and the remaining histidine (His46) must remain as it was for a delocalized binuclear Cu(A) center, or if valence delocalization persists, the hyperfine coupling for one copper must markedly diminish while the overall spin distribution on the CuSSCu core is preserved.

Catalytic function and local proton structure at the type 2 copper of nitrite reductase: the correlation of enzymatic pH dependence, conserved residues, and proton hyperfine structure.

Electron nuclear double resonance (ENDOR) of protons at Type 2 and Type 1 cupric active sites correlates with the enzymatic pH dependence, the mutation of nearby conserved, nonligating residues, and electron transfer in heterologously expressed Rhodobacter sphaeroides nitrite reductase. Wild-type enzyme showed a pH 6 activity maximum but no kinetic deuterium isotope effect, suggesting protons are not transferred in the rate-limiting step of nitrite reduction. However, protonatable Asp129 and His287, both located near the Type 2 center, modulated enzyme activity. ENDOR of the wild-type Type 2 center at pH 6.0 revealed an exchangeable proton with large hyperfine coupling. Dipolar distance estimates indicated that this proton was 2.50-2.75 or 2.25-2.45 A from Type 2 copper in the presence or absence of nitrite, respectively. This proton may provide a properly oriented hydrogen bond to enhance water formation upon nitrite reduction. This proton was eliminated at pH 5.0 and showed a diminished coupling at pH 7.5. Mutations of Asp129 and His287 reduced enzyme activity and altered the exchangeable proton hyperfine spectra. Mutation of Asp129 prevented a pH-dependent change at the Type 1 Cys167 ligand as observed by Cys C(beta) proton ENDOR, implying there is a Type 2 and pH-dependent alteration of the Type 1 center. Mutation of the Type 1 center ligand Met182 to Thr and mutation of Asp129 increased the activation energy for nitrite reduction. Involvement of both the Type 1 center and Asp129 in modulating activation energy shows that electron transfer from the Type 1 center to a nitrite-ligated Type 2 center is rate-limiting for nitrite reduction. Mutation of Ile289 to Ala and Val caused minor perturbation to enzyme activity, but as detected by ENDOR, allowed formate binding. Thus, bulky Ile289 may exclude non-nitrite ligands from the Type 2 active site.

Characterization of a member of the NnrR regulon in Rhodobacter sphaeroides 2.4.3 encoding a haem-copper protein.

Upstream of the nor and nnrR cluster in Rhodobacter sphaeroides 2.4.3 is a previously uncharacterized gene that has been designated nnrS. nnrS is only expressed when 2.4.3 is grown under denitrifying conditions. Expression of nnrS is dependent on the transcriptional regulator NnrR, which also regulates expression of genes required for the reduction of nitrite to nitrous oxide, including nirK and nor. Deletion analysis indicated the sequence 5'-TTGCG(N4)CACAA-3', which is similar to sequences found in nirK and nor, is required for nnrS expression. Mutation of this sequence to the consensus Fnr-binding sequence by changing two bases in each half site caused nnrS expression to become nitrate independent. Inactivation of nnrS did not affect nitric oxide metabolism, nor did it affect expression of any of the genes involved in nitric oxide metabolism. However, taxis towards nitrate and nitrite was affected by nnrS inactivation. Purification of a histidine-tagged NnrS demonstrated that NnrS is a haem- and copper-containing membrane protein. Genes encoding putative orthologues of NnrS are sometimes but not always found in bacteria encoding nitrite and/or nitric oxide reductase.