EPR spectra of ferric cytochromes c' from five strains of Achromobacter xylosoxidans at low temperature and their temperature dependence

The EPR spectra at low temperature (6 K) and their temperature dependence (10-93 K) for five ferric cytochromes c' isolated from chemoheterotrophic bacteria, Achromobacter xylosoxidans NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, GIFU 1048, GIFU 1051, and GIFU 1764 are reported. The EPR spectral results indicate that the ground state of the heme iron(III) of cytochromes c' from these chemoheterotrophic bacteria can appear to be in an admixed spin state which consists of predominant S = 5/2 with a slight S = 3/2 character. The EPR spectra were compared with those for ferric cytochromes c' from photosynthetic bacteria and the other ferric hemoproteins.     

Spectral properties of Achromobacter xylosoxidans cytochromes c' and their NO complexes.

Cytochromes c' have been isolated from six strains of Achromobacter xylosoxidans: NCIB 11015 (formerly Alcaligenes sp. NCIB 11015), GIFU 543, 1048, 1051, 1055 and 1764. They are dimeric proteins with more positive redox potentials than those of cytochromes c' from phototrophic bacteria at neutral pH. The electronic absorption, EPR and MCD spectra on NO-ferrous cytochromes c' at physiological pH showed that the major part of the heme-iron of nitrosylheme was penta-coordinated. The EPR spectral results indicated that the ground state of the heme-iron of ferric cytochromes c' appears to be in an admixed spin states which consists of predominant high-spin with a slight intermediate-spin character at pH 7.2. These spectra were compared with those for cytochromes c' from phototrophic bacteria and the other hemoproteins.     

Purification and some properties of cytochrome c' from a strain of Achromobacter xylosoxidans

Cytochrome c' was crystallized from Achromobacter xylosoxidans GIFU 543. The cytochrome was a basic protein and its molecular weight was 28,000. The pyridine ferrohemochrome showed absorption peaks at 415, 521, and 551 nm. The absorption spectra of the oxidized and reduced forms at neutral pH were almost the same as those of other cytochromes c' reported already. The reduced cytochrome c' reacted with CO and NO, and the NO complex showed a characteristic absorption spectrum. The midpoint redox potential of the hemoprotein was measured to be + 110 mV at pH 7.2.     

Identification of heme axial ligands of cytochrome c' from Alcaligenes sp. N.C.I.B. 11015

The spectral properties of both ferric and ferrous cytochromes c' from Alcaligenes sp. N.C.I.B. 11015 are reported. The EPR spectra at 77 K and the electronic, resonance Raman, CD and MCD spectra at room temperature have been compared with those of the other cytochromes c' and various hemoproteins. In the ferrous form, all the spectral results at physiological pH strongly indicated that the heme iron(II) is in a high-spin state. In the ferric form, the EPR and electronic absorption spectra were markedly dependent upon pH. EPR and electronic spectral results suggested that the ground state of heme iron(III) at physiological pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state. Under highly alkaline conditions, identification of the axial ligands of heme iron(III) was attempted by crystal field analysis of the low-spin EPR g values. Upon the addition of sodium dodecyl sulfate to ferric and ferrous cytochrome c', the low-spin type spectra were induced. The heme environment of this low-spin species is also discussed.     

Spectral properties of nitric oxide complex of cytochrome c' from Rhodopseudomonas capsulata B100

The spectral properties for NO complexes of ferric and ferrous cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata B100 are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the NO complexes of the other cytochromes c' and horse heart cytochrome c. The NO-ferrous cytochrome c' would be a mixture of NO complexes with six- and five-coordinate nitrosylheme, suggesting that the heme-iron to histidine bond in the ferrous cytochrome c' is more stable than that from chemoheterotrophic bacteria. The reaction product of ferric cytochrome c' with NO exhibited the spectra similar to NO-ferric derivatives of the other hemoproteins, which indicates the formation of NO-ferric cytochrome c'.     

Resonance Raman studies of cytochrome c' support the binding of NO and CO to opposite sides of the heme: implications for ligand discrimination in heme-based sensors

Resonance Raman (RR) studies have been conducted on Alcaligenes xylosoxidans cytochrome c', a mono-His ligated hemoprotein which reversibly binds NO and CO but not O(2). Recent crystallographic characterization of this protein has revealed the first example of a hemoprotein which can utilize both sides of its heme (distal and proximal) for binding exogenous ligands to its Fe center. The present RR investigation of the Fe coordination and heme pocket environments of ferrous, carbonyl, and nitrosyl forms of cytochrome c' in solution fully supports the structures determined by X-ray crystallography and offers insights into mechanisms of ligand discrimination in heme-based sensors. Ferrous cytochrome c' reacts with CO to form a six-coordinate heme-CO complex, whereas reaction with NO results in cleavage of the proximal linkage to give a five-coordinate heme-NO adduct, despite the relatively high stretching frequency (231 cm(-1)) of the ferrous Fe-N(His) bond. RR spectra of the six-coordinate CO adduct indicate that CO binds to the Fe in a nonpolar environment in line with its location in the hydrophobic distal heme pocket. On the other hand, RR data for the five-coordinate NO adduct suggest a positively polarized environment for the NO ligand, consistent with its binding close to Arg 124 on the opposite (proximal) side of the heme. Parallels between certain physicochemical properties of cytochrome c' and those of heme-based sensor proteins raise the possibility that the latter may also utilize both sides of their hemes to discriminate between NO and CO binding.     

The pH-dependent changes of intramolecular electron transfer on copper-containing nitrite reductase.

Electron transfer over 12.6 A from the type 1 copper (T1Cu) to the type 2 copper (T2Cu) was investigated in the copper-containing nitrite reductases from two denitrifying bacteria (Alcaligenes xylosoxidans GIFU 1051 and Achromobacter cycloclastes IAN 1013), following pulse radiolytical reduction of T1Cu. In the presence of nitrite, the rate constant for the intramolecular electron transfer of the enzyme from A. xylosoxidans decreased 1/2 fold to 9 x 10(2) s-1 (20 degrees C, pH 7.0) as compared to that for the same process in the absence of nitrite. However, the rate constant increased with decreasing pH to become the same (2 x 10(3) s-1) as that in the absence of nitrite at pH 6.0. A similar result was obtained for the enzyme from A. cycloclastes. The pH profiles of the two enzymes in the presence of nitrite are almost the same as that of the enzyme activity of nitrite reduction. This suggests that the intramolecular electron transfer process is closely linked to the following process of catalytic reduction of nitrite. The difference in redox potential (DeltaE) of T2Cu minus T1Cu was calculated from equilibrium data for the electron transfer. The pH-dependence of DeltaE was in accord with the equation: DeltaE = DeltaE(0)+0.058 log (Kr[H+]+[H+]2)/(K(0)+[H+]), where K(r) and K(0) are the proton dissociation constants for the oxidized and reduced states of T2Cu, respectively. These results raise the possibility that amino acid residues linked by the redox of T2Cu play important roles in the enzyme reaction, being located near T2Cu.     

Amino acid sequences of cytochrome c-554(548) and cytochrome c'' from a halophilic denitrifying bacterium of the genus Paracoccus.

The amino acid sequences of the cytochromes c-554(548) and c' from the moderately halophilic bacterium Paracoccus sp., I.A.M. 203 (= A.T.C.C. 12084, N.C.I.B. 8669) have been determined. Cytochrome c-554(548) consists of a single polypeptide chain of 83 residues, and dimerizes strongly. The most similar protein of known sequence is the N-terminal half of the dihaem cytochrome c4, and other related proteins include the cytochrome c-554(547) of Thiobacillus neapolitanus and the cytochrome c-553 of Desulfovibrio vulgaris. Cytochrome c', which has a single polypeptide chain of 132 residues, is similar in sequence to cytochromes c' from phototrophic and denitrifying bacteria, but only shows about 36% sequence identity to the most similar protein of known sequence. Both of the Paracoccus proteins have a considerable excess of acidic amino acid side chains over basic ones, and a higher proportion of their basic amino acids is arginine than is usual in cytochromes c. Both these characteristics seem to be adaptations to increase the stability of the proteins in an environment of high ionic strength. Detailed evidence for the amino acid sequences of the proteins has been deposited as Supplementary Publication 50140 (24 pp.) at the British Library (Lending Division), Boston Spa, Yorkshire LS23 7BQ, U.K. from which copies are available on prepayment.     

Spectroscopic studies of Rhodobacter capsulatus cytochrome c' in the isolated state and in intact cells.

Ferricytochrome c' from Rhodobacter capsulatus was investigated by 1H-NMR, EPR and optical spectroscopies. A haem-linked ionisation, occurring with a pKa of 8.4 at 25 degrees C, was observed and assigned to the ionisation of the axial histidine ligand by comparison with data for related proteins. At pH values below this pKa the spin-state of the haem Fe3+ is shown to be a quantum mechanically admixed S = 3/2, 5/2 state. Above the pKa the Fe3+ is high-spin. EPR studies of intact cells grown photoheterotrophically reveal that in situ cytochrome c' exists largely in the ferrous state. Upon the addition of [Fe(CN)6]3- the protein becomes oxidised and EPR spectra reveal that the Fe3+ spin-state is a quantum mechanically admixed S = 3/2, 5/2 state. These data indicate that the unusual spin-state of ferricytochrome c' is not a consequence of changes to the protein on its isolation, as had been suggested previously. They also indicate that in situ cytochrome c' is located in an environment with a pH less than 7.     

Cytochromes: Reactivity of the "dark side" of the heme

Ligand binding to the heme distal side is a paradigm of heme-protein biochemistry, the proximal axial ligand being in most cases a His residue. NO binds to the ferrous heme-Fe-atom giving rise to hexa-coordinated adducts (as in myoglobin and hemoglobin) with His and NO as proximal and distal axial ligands, respectively, or to penta-coordinated adducts (as in soluble guanylate cyclase) with NO as the axial distal ligand. Recently, the ferrous derivative of Alcaligenes xylosoxidans cytochrome c' (Axcyt c') and of cardiolipin-bound horse heart cytochrome c (CL-hhcyt c) have been reported to bind NO to the "dark side" of the heme (i.e., as the proximal axial ligand) replacing the endogenous ligand His. Conversely, CL-free hhcyt c behaves as ferrous myoglobin by binding NO to the heme distal side, keeping His as the proximal axial ligand. Moreover, the ferrous derivative of CL-hhcyt c binds CO at the heme distal side, the proximal axial ligand being His. Furthermore, CL-hhcyt c shows peroxidase activity. In contrast, CL-free hhcyt c does not bind CO and does not show peroxidase activity. This suggests that heme-proteins may utilize both sides of the heme for ligand discrimination, which appears to be modulated allosterically. Here, structural and functional aspects of NO binding to ferrous Axcyt c' and (CL-)hhcyt c are reviewed.     

Spectral properties of cytochrome c' from Rhodopseudomonas capsulata B100 and its CO complex

The spectral properties of cytochrome c' from photosynthetic bacterium Rhodopseudomonas capsulata (= Rhodobacter capsulatus) B100 and its CO complex are reported. The electronic absorption, MCD, and EPR spectra have been compared with those of the other cytochromes c' and horse heart cytochrome c. EPR and electronic spectral results for the ferric cytochrome c' suggest that the ground state of heme-iron(III) at neutral pH consists of a quantum mechanical admixture of an intermediate-spin and a high-spin state and that at pH 11.0 is in a high-spin state. In the MCD spectrum of the CO-ferrous cytochrome c', the MCD intensity in the Soret band region was much higher than that of CO complexes of hemoproteins with a protoheme. The differences in a stereochemistry of the sixth-coordination position is discussed.     

Cytochrome c' from Paracoccus denitrificans: spectroscopic studies consistent with a role for the protein in nitric oxide metabolism

Cytochrome c' was purified from the denitrifying bacterium Paracoccus denitrificans and the interaction of the protein with nitric oxide was examined spectroscopically. Two distinct types of haem-nitrosyl electronic absorption spectrum were observed, which were dependent upon [NO]. When cytochrome c' was saturated with NO, alpha and beta bands were centred at 562 nm and 530 nm, whereas with sub-saturating concentrations of NO the alpha and beta bands were red-shifted to 578 nm and 542 nm respectively. Further spectroscopic analysis showed that purified cytochrome c', added to suspensions of P. denitrificans, is able to complex with the NO which is formed as a freely diffusible intermediate of denitrification. In the presence of added NO-3 or NO-2, 40-60% of Fe(II)-cytochrome c' forms a 6-coordinate haem-nitrosyl complex. In the absence of nitrogen oxyanions or NO whole denitrifying cells are able to remove the NO from a Fe(II)-cytochrome c'-NO complex. These findings support the hypothesis that the physiological function of this enigmatic cytochrome involves the reversible binding of nitric oxide.     

Amino acid sequence of cytochrome c' from the purple photosynthetic bacterium Rhodospirillum rubrum S1.

The amino acid sequence of cytochrome c' from the purple photosynthetic bacterium Rhodospirillum rubrum S1 has been determined and is consistent with homology to cytochrome c' from the nonphotosynthetic bacterium Alcaligenes sp. NCIB 11015. There is 29% identity in the chosen alignment of these two proteins. R. rubrum cytochrome c' is composed of a single peptide chain of 126 amino acid residues with a single heme covalently bound near the COOH terminus. There is no sequence similarity to mitochondrial cytochrome c, except at the heme binding site.     

Unprecedented proximal binding of nitric oxide to heme: implications for guanylate cyclase

Microbial cytochromes c' contain a 5-coordinate His-ligated heme that forms stable adducts with nitric oxide (NO) and carbon monoxide (CO), but not with dioxygen. We report the 1.95 and 1.35 A resolution crystal structures of the CO- and NO-bound forms of the reduced protein from Alcaligenes xylosoxidans. NO disrupts the His-Fe bond and binds in a novel mode to the proximal face of the heme, giving a 5-coordinate species. In contrast, CO binds 6-coordinate on the distal side. A second CO molecule, not bound to the heme, is located in the proximal pocket. Since the unusual spectroscopic properties of cytochromes c' are shared by soluble guanylate cyclase (sGC), our findings have potential implications for the activation of sGC induced by the binding of NO or CO to the heme domain.     

Enzymatic removal of nitric oxide catalyzed by cytochrome c' in Rhodobacter capsulatus

Cytochrome c' from Rhodobacter capsulatus has been shown to confer resistance to nitric oxide (NO). In this study, we demonstrated that the amount of cytochrome c' synthesized for buffering of NO is insufficient to account for the resistance to NO but that the cytochrome-dependent resistance mechanism involves the catalytic breakdown of NO, under aerobic and anaerobic conditions. Even under aerobic conditions, the NO removal is independent of molecular oxygen, suggesting cytochrome c' is a NO reductase. Indeed, we have measured the product of NO breakdown to be nitrous oxide (N(2)O), thus showing that cytochrome c' is behaving as a NO reductase. The increased resistance to NO conferred by cytochrome c' is distinct from the NO reductase pathway that is involved in denitrification. Cytochrome c' is not required for denitrification, but it has a role in the removal of externally supplied NO. Cytochrome c' synthesis occurs aerobically and anaerobically but is partly repressed under denitrifying growth conditions when other NO removal systems are operative. The inhibition of respiratory oxidase activity of R. capsulatus by NO suggests that one role for cytochrome c' is to maintain oxidase activity when both NO and O(2) are present.     

The amino acid sequence of cytochrome c′ from Alcaligenes sp. N.C.I.B. 11015

The amino acid sequence of the cytochrome c' from Alcaligenes sp. N.C.I.B. 11015 (Iwasaki's ;Pseudomonas denitrificans') has been determined. This organism is the only non-photosynthetic bacterium in which the protein has been found. The protein consists of a single polypeptide chain of 127 residues, with a single haem covalently attached to two cysteines. Unlike normal cytochromes c, the haem attachment site is very close to the C-terminus. The amino acid sequence around the haem attachment site is very similar to that of Chromatium vinosum D cytochrome c'. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50022 at the British Library (Lending Division), (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Accessibility of the distal heme face, rather than Fe-His bond strength, determines the heme-nitrosyl coordination number of cytochromes c': evidence from spectroscopic studies

The heme coordination chemistry and spectroscopic properties of Rhodobacter capsulatus cytochrome c' (RCCP) have been compared to data from Alcaligenes xylosoxidans (AXCP), with the aim of understanding the basis for their different reactivities with nitric oxide (NO). Whereas ferrous AXCP reacts with NO to form a predominantly five-coordinate heme-nitrosyl complex via a six-coordinate intermediate, RCCP forms an equilibrium mixture of six-coordinate and five-coordinate heme-nitrosyl species in approximately equal proportions. Ferrous RCCP and AXCP both exhibit high Fe-His stretching frequencies (227 and 231 cm(-)(1), respectively), suggesting that factors other than the Fe-His bond strength account for their differences in heme-nitrosyl coordination number. Resonance Raman spectra of ferrous-nitrosyl RCCP confirm the presence of both five-coordinate and six-coordinate heme-NO complexes. The six-coordinate heme-nitrosyl of RCCP exhibits a fairly typical Fe-NO stretching frequency (569 cm(-)(1)), in contrast to the relatively high value (579 cm(-)(1)) of the AXCP six-coordinate heme-nitrosyl intermediate. It is proposed that NO experiences greater steric hindrance in binding to the distal face of AXCP, as compared to RCCP, leading to a more distorted Fe-N-O geometry and an elevated Fe-NO stretching frequency. Evidence that RCCP has a more accessible distal coordination site than in AXCP stems from the fact that ferric RCCP readily forms a heme complex with exogenous imidazole, whereas AXCP does not. A model is proposed in which distal heme-face accessibility, rather than the proximal Fe-His bond strength, determines the heme-nitrosyl coordination number in cytochromes c'.     

Six- to five-coordinate heme-nitrosyl conversion in cytochrome c' and its relevance to guanylate cyclase

The 5-coordinate ferrous heme of Alcaligenes xylosoxidans cytochrome c' reacts with NO to form a 6-coordinate nitrosyl intermediate (lambdaSoret at 415 nm) which subsequently converts to a 5-coordinate nitrosyl end product (lambdaSoret at 395 nm) in a rate-determining step. Stopped-flow measurements at pH 8.9, 25 degrees C, yield a rate constant for the formation of the 6-coordinate nitrosyl adduct, k(on) = (4.4 +/- 0.5) x 10(4) M(-1) x s(-1), which is 3-4 orders of magnitude lower than the values for other pentacoordinate ferrous hemes and is consistent with NO binding within the sterically crowded distal heme pocket. Resonance Raman measurements of the freeze-trapped 6-coordinate nitrosyl intermediate reveal an unusually high Fe-NO stretching frequency of 579 cm(-1), suggesting a distorted Fe-N-O coordination geometry. The rate of 6- to 5-coordinate heme nitrosyl conversion is also dependent upon NO concentration, with a rate constant, k(6-5) = (8.1 +/- 0.7) x 10(3) M(-1) x s(-1), implying that an additional molecule of NO is required to form the 5c-NO adduct. Since crystallographic studies have shown that the 5-coordinate nitrosyl complex of cytochrome c' binds NO to the proximal (rather than distal) face of the heme, the NO dependence of the 6- to 5-coordinate NO conversion supports a mechanism in which the weakened His ligand, as well as the distally bound NO, is displaced by a second NO molecule which attacks and is retained in the proximal coordination position. The fact that a dependent 6- to 5-coordinate nitrosyl conversion has been previously reported for soluble guanylate cyclase suggests that the mechanism of Fe-His bond cleavage may be similar to that of cytochrome c' and strengthens the recent proposal that both proteins exhibit proximal NO binding in their 5-coordinate nitrosyl adducts.     

Use of maleic acid by mixed cultures of microorganisms

In a mixed batch culture, Alcaligenes xylosoxidans subsp. xylosoxidans 260 transformed maleic acid into malic acid. Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid. Both bacterial cultures dissociated with the formation of R, S, and M forms. At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A. xylosoxidans and Bacillus subtilis. At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively. Association of bacteria A. xylosoxidans and B. subtilis was not stable under flow conditions water.