pH dependence of redox potential in c-type cytochromes

Abstract

The midpoint redox potential (Em) of mitochondrial cytochrome c is independent of pH below pH9 (Rodkey & Ball, 1950). For several bacterial homologues of cytochrome c, however, Em is markedly influenced by pH between pH5 and 9; in the case of Pseudomonas aeruginosa cytochrome ᶜ551 , the pH dependence has been interpreted as being due to the redox state dependent ionisation of a haem propionic acid substituent, which has a pK of 6.3 in the oxidised form of of the cytochrome and 7.2 in the reduced form (Moore et al., 1980). The redox state imposed separation of the propionic acid pK values was suggested to occur through the electrostatic influence of the haem, which carries a net charge of +1 in the ferricytochrome but 0 net charge in the ferrocytochrome.In an extension of the studies initiated with P. aeruginosa cytochrome ᶜ551, the bulk of the work described in this thesis deals with the effect of pH on Em for a closely related cytochrome, Pseudomonas stutzeri 221 cytochrome ᶜ551. The P. stutzeri cytochrome shows a similar pattern of pH dependence of Em in that two pKs (pK₀ and pKᵣ) influence Em below pH9; however, pK₀ and pKᵣ occur nearly one pH unit higher (pK₀ = 7.6, pKᵣ = 8.3) than in P. aeruginosa cytochrome ᶜ551. Using high resolution ¹H NMR it was possible to show that haem propionic acid-7 ionises at pK = 7.6 in P. stutzeri ferricytochrome ᶜ551 and also that a histidine residue, His47, is deprotonated with pK = 7.6 in the ferricytochrome and pK = 8.3 in the ferrocytochrome. The pK of propionic acid-7 in the ferrocytochrome could not be measured by NMR and so it was not possible to demonstrate directly that its pK is redox state dependent. However, by chemically modifying His47 with ethoxyformic anhydride, which prevents the histidine from ionising between pH5 and 9, it was shown that the redox potential of P. stutzeri cytochrome was still pH dependent, implying that the redox state dependent ionisation of propionic acid-7 must indeed contribute to the fall in Em at alkaline pH. Since propionic acid-7 and His47 appear to ionise with exactly the same values of pK₀ and pKᵣ in the unmodified cytochrome, and since the propionic acid pKs are lowered by nearly 2 pH units upon modification of His47 with ethoxyformic anhydride, it was deduced that the two groups must interact with each other in a manner that raises the pKs of both species and allows their conjoint ionisation. A hydrogen bonding scheme is proposed in which propionic acid-7 and His47 both exist in a partially charged state at low pH.It was predicted that propionic acid-7 will ionise with redox state dependent pKs in all other cytochromes ᶜ551, and that the nature of the amino acid at sequence position 47 will be important in determining the values of pK₀ and pKᵣ. In this respect it was shown that P. stutzeri 224 and P. mendocina cytochromes ᶜ551, which both contain histidine at sequence position 47, have similar Em versus pH curves and pKᵣ/pKᵣ values to the P. stutzeri 221 cytochrome. NMR data for P. mendocina cytochrome ᶜ551 confirmed that propionic acid-7, and His47, ionise in this cytochrome also. However, P. denitrificans and Azotobacter vinelandii cytochromes ᶜ551 show only very slight pH dependence of redox potential below pH9 (i.e. pK₀ and pKᵣ very close together), a pattern which was not predictable in either case simply from consideration of the identity of the amino acid occurring at position 47 in the sequence.The Em versus pH curve was also obtained for Chlorobium thiosulphatophilum cytochrome ᶜ551, a cytochrome which is distantly related in sequence to the cytochromes ᶜ551. The general shape of the curve is similar to that observed for the cytochromes ᶜ551, with pK₀ = 6.3 and pKᵣ = 7.1. NMR data showed that a histidine residue is deprotonated with these pKs; this histidine may be sequentially analogous to His47 of P. stutzeri and P. mendocina cytochromes ᶜ551. The NMR data also suggested that a propionic acid substituent ionises. Thus the redox potential of cytochrome ᶜ555 appears to be affected by the same mechanism as in P. stutzeri and P. mendocina cytochromes ᶜ551, despite their diverse origins.