Biomedical subjects

James P Shapleigh

Publications and source records attributed to James P Shapleigh.

17 recordsLinked to original sources

PubMed2007

Selenite-reducing capacity of the copper-containing nitrite reductase of Rhizobium sullae.

Rhizobium sullae strain HCNT1 contains a nitric oxide-producing nitrite reductase of unknown function due to the absence of a complementary nitric oxide reductase. HCNT1 had the ability to grow on selenite concentrations as high as 50 mM, and during growth, selenite was reduced to the less toxic elemental selenium. An HCNT1 mutant lacking nitrite reductase grew poorly in the presence of 5 mM selenite, was unable to grow in the presence of 25 or 50 mM selenite and also showed no evidence of selenite reduction. A naturally occurring nitrite reductase-deficient R. sullae strain, CC1335, also showed little growth on the higher concentrations of selenite. Mobilization of a plasmid containing the HCNT1 gene encoding nitrite reductase into CC1335 increased its resistance to selenite. To confirm that this ability to grow in the presence of high concentrations of selenite correlated with nitrite reductase activity, a new nitrite reductase-containing strain was isolated from the same location where HCNT1 was isolated. This strain was also resistant to high concentrations of selenite. Inactivation of the gene encoding nitrite reductase in this strain increased selenite sensitivity. These data suggest that the nitrite reductase of R. sullae provides resistance to selenite and offers an explanation for the radically truncated denitrification found uniquely in this bacterium.

Bacterial Proteins↗

PubMed2006

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).

Copper↗

PubMed2006

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.

Cytochromes c'↗

PubMed2006

Electron transfer to nitrite reductase of Rhodobacter sphaeroides 2.4.3: examination of cytochromes c2 and cY.

The role of cytochrome c(2), encoded by cycA, and cytochrome c(Y), encoded by cycY, in electron transfer to the nitrite reductase of Rhodobacter sphaeroides 2.4.3 was investigated using both in vivo and in vitro approaches. Both cycA and cycY were isolated, sequenced and insertionally inactivated in strain 2.4.3. Deletion of either gene alone had no apparent effect on the ability of R. sphaeroides to reduce nitrite. In a cycA-cycY double mutant, nitrite reduction was largely inhibited. However, the expression of the nitrite reductase gene nirK from a heterologous promoter substantially restored nitrite reductase activity in the double mutant. Using purified protein, a turnover number of 5 s(-1) was observed for the oxidation of cytochrome c(2) by nitrite reductase. In contrast, oxidation of c(Y) only resulted in a turnover of approximately 0.1 s(-1). The turnover experiments indicate that c(2) is a major electron donor to nitrite reductase but c(Y) is probably not. Taken together, these results suggest that there is likely an unidentified electron donor, in addition to c(2), that transfers electrons to nitrite reductase, and that the decreased nitrite reductase activity observed in the cycA-cycY double mutant probably results from a change in nirK expression.

Amino Acid Sequence↗

PubMed2006

Assessing the impact of denitrifier-produced nitric oxide on other bacteria.

A series of experiments was undertaken to learn more about the impact on other bacteria of nitric oxide (NO) produced during denitrification. The denitrifier Rhodobacter sphaeroides 2.4.3 was chosen as a denitrifier for these experiments. To learn more about NO production by this bacterium, NO levels during denitrification were measured by using differential mass spectrometry. This revealed that NO levels produced during nitrate respiration by this bacterium were in the low muM range. This concentration of NO is higher than that previously measured in denitrifiers, including Achromobacter cycloclastes and Paracoccus denitrificans. Therefore, both 2.4.3 and A. cycloclastes were used in this work to compare the effects of various NO levels on nondenitrifying bacteria. By use of bacterial overlays, it was found that the NO generated by A. cycloclastes and 2.4.3 cells during denitrification inhibited the growth of both Bacillus subtilis and R. sphaeroides 2.4.1 but that R. sphaeroides 2.4.3 caused larger zones of inhibition in the overlays than A. cycloclastes. Both R. sphaeroides 2.4.3 and A. cycloclastes induced the expression of the NO stress response gene hmp in B. subtilis. Taken together, these results indicate that there is variability in the NO concentrations produced by denitrifiers, but, irrespective of the NO levels produced, microbes in the surrounding environment were responsive to the NO produced during denitrification.

Achromobacter cycloclastes↗

PubMed2005

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.

Binding Sites↗

PubMed2005

Expression of nitrite and nitric oxide reductases in free-living and plant-associated Agrobacterium tumefaciens C58 cells.

A number of the bacteria that form associations with plants are denitrifiers. To learn more about how the association with plants affects expression of denitrification genes, the regulation of nitrite and nitric oxide reductases was investigated in Agrobacterium tumefaciens. Analysis of free-living cells revealed that expression of the genes encoding nitrite and nitric oxide reductases, nirK and nor, respectively, requires low-oxygen conditions, nitric oxide, and the transcriptional regulator NnrR. Expression of nor was monitored in plant-associated bacteria using nor-gfp fusion expression. In root association experiments, only a small percentage of the attached cells were fluorescent, even when they were incubated under a nitrogen atmosphere. Inactivation of nirK had no significant effect on the ability of A. tumefaciens to bind to plant roots regardless of the oxygen tension, but it did decrease the occurrence of root-associated fluorescent cells. When wild-type cells containing the gfp fusion were infiltrated into leaves, most cells eventually became fluorescent. The same result was obtained when a nirK mutant was used, suggesting that nitric oxide activated nor expression in the endophytic bacteria. Addition of a nitric oxide synthase inhibitor to block nitric oxide generation by the plant prevented gfp expression in infiltrated nitrite reductase mutants, demonstrating that plant-derived nitric oxide can activate nor expression in infiltrated cells.

Agrobacterium tumefaciens↗

PubMed2005

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.

Bacterial Proteins↗

PubMed2004

Denitrification genes regulate Brucella virulence in mice.

Brucella is the causative agent of the zoonotic disease brucellosis, which is endemic in many parts of the world. Genome sequencing of B. suis and B. melitensis revealed that both are complete denitrifiers. To learn more about the role of denitrification in these animal pathogens, a study of the role of denitrification in the closely related B. neotomae was undertaken. In contrast to B. suis and B. melitensis, it was found that B. neotomae is a partial denitrifier that can reduce nitrate to nitrite but no further. Examination of the B. neotomae genome showed that a deletion in the denitrification gene cluster resulted in complete loss of nirV and the partial deletion of nirK and nnrA. Even though the nor operon is intact, a norC-lacZ promoter fusion was not expressed in B. neotomae. However, the norC-lacZ fusion was expressed in the related denitrifier Agrobacterium tumefaciens, suggesting that the lack of expression in B. neotomae is due to inactivation of NnrA. A narK-lacZ promoter fusion was found to exhibit nitrate-dependent expression consistent with the partial denitrifier phenotype. Complementation of the deleted region in B. neotomae by using nirK, nirV, and nnrA from B. melitensis restored the ability of B. neotomae to reduce nitrite. There was a significant difference in the death of IRF-1-/- mice when infected with B. neotomae containing nirK, nirV, and nnrA and those infected with wild-type B. neotomae. The wild-type strain killed all the infected mice, whereas most of the mice infected with B. neotomae containing nirK, nirV, and nnrA survived.

Adaptation, Physiological↗

PubMed2003

Site-directed mutagenesis of NnrR: a transcriptional regulator of nitrite and nitric oxide reductase in Rhodobacter sphaeroides.

NnrR, a transcriptional activator and member of the CRP/FNR family of regulators, is responsible for controlling the expression of a number of denitrification genes in Rhodobacter sphaeroides 2.4.3. The apparent effector for NnrR is nitric oxide, and in its presence NnrR activates expression of the nirK gene and the nor operon, encoding nitrite reductase and nitric oxide reductase, respectively. Whether nitric oxide directly interacts with NnrR to activate transcription is unknown. Other denitrifiers carry putative orthologs of NnrR. To gain insight into NnrR function, a number of conserved residues were mutagenized. The impact of these changes on NnrR function was assessed by monitoring expression of a nirK-lacZ fusion. In this way a region spanning from Tyr93 to Cys103 that contains residues critical for NnrR activity was identified.

Bacterial Proteins↗

PubMed2003

Spectroscopic studies of the Met182Thr mutant of nitrite reductase: role of the axial ligand in the geometric and electronic structure of blue and green copper sites.

A combination of spectroscopic methods and density functional calculations has been used to describe the electronic structure of the axial mutant (Met182Thr) of Rhodobacter sphaeroides nitrite reductase in which the axial methionine has been changed to a threonine. This mutation results in a dramatic change in the geometric and electronic structure of the copper site. The electronic absorption data imply that the type 1 site in the mutant is like a typical blue copper site in contrast to the wild-type site, which is green. Similar ligand field strength in the mutant and the wild type (from MCD spectra) explains the similar EPR parameters for very different electronic structures. Resonance Raman shows that the Cu-S(Cys) bond is stronger in the mutant relative to the wild type. From a combination of absorption, CD, MCD, and EPR data, the loss of the strong axial thioether (present in the wild-type site) results in an increase of the equatorial thiolate-Cu interaction and the site becomes less tetragonal. Spectroscopically calibrated density functional calculations were used to provide additional insight into the role of the axial ligand. The calculations reproduce well the experimental ground-state bonding and the changes in going from a green to a blue site along this coupled distortion coordinate. Geometry optimizations at the weak and strong axial ligand limits show that the bonding of the axial thioether is the key factor in determining the structure of the ground state. A comparison of plastocyanin (blue), wild-type nitrite reductase (green), and the Met182Thr mutant (blue) sites enables evaluation of the role of the axial ligand in the geometric and electronic structure of type 1 copper sites, which can affect the electron-transfer properties of these sites.

Bacterial Proteins↗

PubMed2003

Use of a green fluorescent protein-based reporter fusion for detection of nitric oxide produced by denitrifiers.

To determine if green fluorescent protein could be used as a reporter for detecting nitric oxide production, gfp was fused to nnrS from Rhodobacter sphaeroides 2.4.3. nnrS was chosen because its expression requires nitric oxide. The presence of the fusion in R. sphaeroides 2.4.3 resulted in a significant increase in fluorescent intensity of the cells, but only when nitrite reductase was active. Cells lacking nitrite reductase activity and consequently the ability to generate nitric oxide were only weakly fluorescent when grown under denitrification-inducing conditions. One of the R. sphaeroides strains unable to generate nitric oxide endogenously was used as a reporter to detect exogenously produced nitric oxide. Incubation of this strain with sodium nitroprusside, a nitric oxide generator, significantly increased its fluorescence intensity. Mixing of known denitrifiers with the reporter strain also led to significant increases in fluorescence intensity, although the level varied depending on the denitrifier used. The reporter was tested on unknown isolates capable of growing anaerobically in the presence of nitrate, and one of these was able to induce expression of the fusion. Analysis of the 16S rRNA gene sequence of this isolate placed it within the Thauera aromatica subgroup, which is known to contain denitrifiers. These experiments demonstrate that this green fluorescent protein-based assay provides a useful method for assessing the ability of bacteria to produce nitric oxide.

Bacterial Proteins↗

PubMed2002

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.

Amino Acid Substitution↗

PubMed2002

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.

Bacterial Proteins↗

PubMed2002

Taxis response of various denitrifying bacteria to nitrate and nitrite.

The taxis response of Rhodobacter sphaeroides 2.4.1 and 2.4.3, Rhodopseudomonas palustris, and Agrobacterium tumefaciens to nitrate and nitrite was evaluated by observing the macroscopic behavior of cells suspended in soft agar and incubated under various conditions. R. sphaeroides 2.4.3, which is capable of both nitrate and nitrite reduction, showed a taxis response to both nitrate and nitrite. R. sphaeroides 2.4.1, which contains nitrate reductase but not nitrite reductase, did not show a taxis response towards either nitrogen oxide. Insertional inactivation of the nitrite reductase structural gene or its transcriptional regulator, NnrR, in strain 2.4.3 caused a loss of a taxis response towards both nitrate and nitrite. An isolate of 2.4.1 carrying a copy of the nitrite reductase gene from 2.4.3 showed a taxis response to both nitrogen oxides. The taxis response of 2.4.3 was observed under anaerobic conditions, suggesting that the taxis response was due to nitrate and nitrite respiration, not to inhibition of oxygen respiration by respiration of nitrogen oxides. Strain 2.4.3 showed a taxis response to nitrate and nitrite under photosynthetic and aerobic conditions. Changing the carbon source in the culture medium caused an unexpected subtle shift in the taxis response of 2.4.3 to nitrite. A taxis response to nitrogen oxides was also observed in R. palustris and A. tumefaciens. R. palustris exhibited a taxis response to nitrite but not to nitrate, while A. tumefaciens exhibited a response to both compounds.

Agrobacterium tumefaciens↗

PubMed2002

Involvement of the PrrB/PrrA two-component system in nitrite respiration in Rhodobacter sphaeroides 2.4.3: evidence for transcriptional regulation.

Rhodobacter sphaeroides strain 2.4.3 is capable of diverse metabolic lifestyles, including denitrification. The regulation of many Rhodobacter genes involved in redox processes is controlled, in part, by the PrrBA two-component sensor-regulator system, where PrrB serves as the sensor kinase and PrrA is the response regulator. Four strains of 2.4.3 carrying mutations within the prrB gene were isolated in a screen for mutants unable to grow anaerobically on medium containing nitrite. Studies revealed that the expression of nirK, the structural gene encoding nitrite reductase, in these strains was significantly decreased compared to its expression in 2.4.3. Disruption of prrA also eliminated the ability to grow both photosynthetically and anaerobically in the dark on nitrite-amended medium. Complementation with prrA restored the wild-type phenotype. The PrrA strain exhibited a severe decrease in both nitrite reductase activity and expression of a nirK-lacZ fusion. Nitrite reductase activity in the PrrA strain could be restored to wild-type levels by using nirK expressed from a heterologous promoter, suggesting that the loss of nitrite reductase activity in the PrrA and PrrB mutants was not due to problems with enzyme assembly or the supply of reductant. Inactivation of prrA had no effect on the expression of the gene encoding NnrR, a transcriptional activator required for the expression of nirK. Inactivation of ccoN, part of the cbb(3)-type cytochrome oxidase shown to regulate the kinase activity of PrrB, also caused a significant decrease in both nirK expression and Nir activity. This was unexpected, since PrrA-P accumulates in the ccoN strain. Together, these results demonstrate that PrrBA plays an essential role in the regulation of nirK.

Bacterial Proteins↗

PubMed2001

Characterization of nirV and a gene encoding a novel pseudoazurin in Rhodobacter sphaeroides 2.4.3.

Sequencing of the region flanking nirK, the gene encoding the copper-containing nitrite reductase in Rhodobacter sphaeroides 2.4.3, has identified two genes whose products could potentially be involved in nitrite reductase expression and activity. One of the genes has been designated nirV. Putative nirV orthologues are found in other denitrifiers, where they are also located downstream of the structural gene for nitrite reductase. The nirV in 2.4.3 is apparently cotranscribed with nirK. Inactivation of nirV had no effect on cell growth, or on nitrite reductase expression or activity. Downstream of nirV and divergently transcribed is a gene, designated ppaZ, encoding a protein with significant similarity to pseudoazurins from other denitrifiers. However, three of the four residues required for binding of the type I copper centre are not conserved in the deduced sequence of the protein in 2.4.3. ppaZ is expressed only when oxygen becomes limiting. ppaZ expression is dependent on both FnrL and NnrR, and a putative binding site for these proteins has been identified. Expression of ppaZ is also dependent on the two-component PrrB/PrrA system. Inactivation of ppaZ had no significant effect on cell growth or on nitrite reductase expression or activity. Expression of a maltose-binding protein-PpaZ fusion indicated that the protein could not bind copper. Examination of the genome of the related bacterium R. sphaeroides 2.4.1 revealed that it encodes ppaZ but not nirV and evidence is presented suggesting that a common ancestor of 2.4.3 and 2.4.1 had both nitrite and nitric oxide reductase activity but as the strains diverged 2.4.1 lost nirK and nirV, making it incapable of nitrite reduction.

Amino Acid Sequence↗