The 10th Nitrogen Cycle Meeting 2004

Purification and characterization of cytochrome c′ from Neisseria meningitidis

W.M. Huston, E.C. Lowe, C.S. Butler, J.W.B. Moir


Cytochrome c′, a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c′ has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c′ from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c′ demonstrated a pH-dependent transition (over the pH range of 6–10) typical of known c′-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)–NO complexes were formed when Fe(II) or Fe(III) cytochrome c′ was sparged with NO gas, whereas an Fe(II)–NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).

  • cytochrome c
  • Neisseria meningitidis
  • nitrosyl complex
  • nitric oxide
  • purification
  • spectroscopy


Cytochrome c′, a cytochrome with a C-terminal, covalently attached haem and unusual magnetic and electronic spectral properties, has been isolated from a variety of denitrifying and photosynthetic α- and β-proteobacteria. The cytochrome has been shown genetically to have a role in the detoxification of NO in Rhodobacter capsulatus and Neisseria meningitidis, although the mechanism of this protection is yet to be determined [13]. The pathogenic Neisseria, N. meningitidis and Neisseria gonorrhoeae, contain genes sharing homology with cytochrome c′, but the protein has not yet been characterized from a pathogen. N. meningitidis is the predominant causative agent for bacterial meningitis and is asymptomatically carried in 10% of the population [4]. A mutant deficient in cytochrome c′ generated in N. meningitidis was demonstrated by Anjum et al. [3] to be more sensitive to NO donors when compared with the wild-type strain, indicating a probable function in protection against NO stress. To examine the function of cytochrome c′ in protection against nitrosative stress, the protein was overexpressed in Escherichia coli, purified and examined spectroscopically.

Materials and methods

The cycP gene encoding cytochrome c′ was amplified by PCR from the N. meningitidis genome for overexpression. As the gene product is predicted to be located attached to the outer membrane, the signal sequence and lipoprotein attachment site were excluded from the overexpression construct by the primer design. The PCR was conducted using the primers cycP1 (5′-tttccatgggaggcagcggaataccttc-3′) and cycP2 (5′-tctgagctcttcagacggcattg-3′). The restriction enzyme sites NcoI and SacI (underlined) were incorporated into the primers to enable cloning of the PCR product. The expression vector pET22b has an N-terminal PelB signal sequence that directs the localization of the expressed protein to the periplasm, essential for the covalent attachment of the porphyrin to the protein. The cycP gene, amplified from N. meningitidis MC58 with Pfu polymerase, was cloned into pET22b, yielding pET22bcycP.

Cytochrome c′ was expressed in the E. coli strain BL21 DE3. The cytochrome c biogenesis operon was supplied in trans on the plasmid pST2 [5] to enable the aerobic expression of c′. The optimal growth and expression conditions for the highest yield of protein were at 37°C with mid-logarithmic induction of cytochrome c′ with 1 mM isopropyl β-D-thiogalactoside followed by 3 h out-growth. Initially, periplasmic extractions of E. coli were attempted; however, most of the cytochrome c′ was not released. (Association with the cytoplasmic membrane has been observed previously for the E. coli overexpression of cytochrome c′ from Rhodopseudomonas palustris [6]). The protein was therefore purified from total extracts of the BL21 DE3 pST2 pET22bcycp strain, which had been lysed by sonication. The protein was purified from these extracts using a variety of chromatographic techniques; this procedure and the resulting yield of pure cytochrome c′ are shown in Figure 1.

Figure 1 Purification of cytochrome c′ from BL21 DE3 pET22bcycp pST2

A flow chart of the purification method for the isolation of pure cytochrome c′ is shown. The purity index and final yields are indicated.

UV–visible spectroscopy was conducted using the Jasco 32 spectrophotometer. Samples were reduced with sodium dithionite (Sigma–Aldrich, St. Louis, MO, U.S.A.) when required. NO was added either in the form of NO gas which was bubbled through a 1 M NaOH solution before bubbling into the sample (Sigma–Aldrich) or using an NO donor; DEA NONOate (diazeniumdiolate; AG Scientific, San Diego, CA, U.S.A.). DEA NONOate has a half-life of 16 min at pH 7.4 and 22°C. EPR spectra were measured using a Bruker EMX spectrometer (X-band, 9.38 GHz) equipped with an ER4112HV liquid helium flow cryostat system. Cytochrome c′ haem spectra were recorded at 10 K, 4.0 G modulation amplitude and 2.0 mW power. Cytochrome c′-NO spectra were recorded at 77 K, 2.0 G modulation amplitude and 10.0 mW power.

Results and discussion

The purified cytochrome c′ was examined spectroscopically as shown in Figure 2. The oxidized protein showed the spectral properties typical of cytochrome c′ with the Soret band at 400 nm, a broad αβ region from approx. 480–560 nm and a peak at 638 nm corresponding to a high-spin feature. The spectrum of the reduced protein showed a shift in the Soret band to 430 nm and a single peak in the αβ region, which had a plateau at 550 nm.

Figure 2 Spectroscopic examination of cytochrome c

The UV–visible spectra of cytochrome c′ (oxidized and reduced) and the resulting Fe(III) c′–NO complex formed when both redox forms were sparged with NO gas is depicted. The Fe(II) c′–NO five-co-ordinate complexes that were formed when each form was incubated with 10 mM DEA NONOate and the six-co-ordinate Fe(II) c′–NO complex formed when Fe(III) cytochrome c′ was supplied with 300 μM DEA NONOate are shown. An inset of the αβ region (465–625 nm) of each of the spectra is shown (×5) enlarged.

The electronic absorption spectra of the ferric cytochrome c′ demonstrated some pH-dependent transitions. The Soret peak had a shoulder, which became apparent and broader at higher pH; this shoulder has been observed previously on the related cytochromes c′ of Rhodospirillum molischianum and Rhodospirillum rubrum [7]. For these related proteins also, it was observed that the high-spin feature at 638 nm increased as the pH was increased, indicating that, at higher pH, there is an increase in the high-spin state of the protein.

c′-type cytochromes bind NO, thus generating characteristic electronic and magnetic spectra. We succeeded in trapping three distinct cytochrome-NO spectra. Ferric cytochrome c′ sparged with NO gas yields a spectrum with the Soret band at 417 nm and distinctive α and β bands at 565 and 530 nm (Figure 2). This is typical of a ferric nitrosyl and is consistent with the absence of an Fe(II)-nitrosyl spectrum in EPR (results not shown). Ferrous cytochrome c′ also formed a ferric cytochrome c′ complex after sparging with NO gas. However, when incubated with the NO donor DEA NONOate, both the ferrous and ferric c′-type cytochromes formed a ferrous cytochrome c′–NO complex that showed spectral features consistent with a five-co-ordinate complex. The Soret band was located at 402 nm; a peak at 480 nm consistent with a five-co-ordinate NO complex and a broad NO αβ region with a plateau between 535 and 570 nm were observed (Figure 2). This formation of the ferrous cytochrome c′–NO complex from the oxidized protein is probably due to a reductive nitrosylation event, which was observed previously for cytochrome c′ from denitrifying bacteria [8]. The reduced cytochrome c′–NO complex was examined by EPR spectroscopy, which gave rise to a typical three-line split signal indicative of a five-co-ordinate ferrous c′–NO complex, consistent with that previously reported for the denitrifying bacteria Achromobacter xylosoxidans and Alcaligenes sp. NCIB 11015 [9,10]. The use of DEA NONOate at a lower concentration when incubated with ferric cytochrome c′ resulted in the formation of what seems likely to be a six-co-ordinate ferrous cytochrome c′ complex, although this remains to be confirmed by EPR spectroscopy.

The present study demonstrates that the cycP gene from N. meningitidis is capable of synthesizing a cytochrome c′ with typical spectroscopic features. Furthermore, the fact that the protein from this source may be suitable for mechanistic studies of NO detoxification is highlighted by our success in trapping three distinct nitrosyl forms of the cytochrome.


We thank S. Turner and J. Cole for the valuable contribution of the pST2 plasmid. J.W.B.M. was supported by grant no. 87/C19243 from BBSRC.


  • The 10th Nitrogen Cycle Meeting 2004: Focused Meeting held at the University of East Anglia, Norwich, U.K., 2–4 September 2004. Edited by C.S. Butler (Newcastle upon Tyne, U.K.) and D.J. Richardson (Norwich, U.K.). Sponsored by the COST (European Cooperation in the field of Scientific and Technical Research) Office and the ESF (European Science Foundation).


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