Binding spectra with cytochrome

Reaction of Cytochrome cIinn with Bis(ferrozine)copper(II) Knowledge of the redox properties of cytochrome c was an encouragement to initiate a kinetics investigation of the reduction of an unusual copper(II) complex species by cyt c11. Ferrozine (5,6-bis(4-sulphonatophenyl)-3-(2-pyridyl)-1,2.4-triazine)286 (see Scheme 7.1), a ligand that had come to prominence as a sensitive spectrophotometric probe for the presence of aqua-Fe(II),19c,287 forms a bis complex with Cu(II) that is square pyramidal, with a water molecule in a fifth axial position, whereas the bis-ferrozine complex of Cu(I) is tetrahedral.286 These geometries are based primarily upon analysis of the UV/visible spectrum. Both complexes are anionic, as for the tris-oxalato complex of cobalt in reaction with cytochrome c (Section 7.3.3.4), the expectation is that the two partners will bind sufficiently strongly in the precursor complex to allow separation of the precursor formation constant from the electron transfer rate constant, from the empirical kinetic data. 

Confirmation of intracellular GSH binding was obtained by H SEFT studies. Figure 3 at 0.5 mM Au the Cys 3CH2 resonance disappears from the spectrum and the marked decrease in intensity of the Gly CH2 resonance indicates non-equivalence, as seen for model reactions. It remains to be seen whether GSH protects oxyhaemo-globin from EtaPAuCl-induced spin-state changes (26) similar to those we have observed with cytochrome c (27). 

Type II substrates are compounds such as nitrogenous bases, with sp2 or sp3 nonbonded electrons. These bind to iron and give rise to a 6-coordinated, low-spin hemoprotein. Such compounds may also be inhibitors of cytochromes P-450. The spectrum shows a peak at 420 to 435 nm and a trough at 390 to 410 nm in the difference spectrum. 

In the reduced state, cytochromes P-450 may also bind certain ligands to give particular difference spectra. The most well known is that which occurs when carbon monoxide binds giving an absorption maximum at 450 nm. A type III spectrum gives two peaks at 430 and 455 nm after binding of certain compounds such as ethyl isocyanide or methylenedioxyphenyl compounds to the reduced enzyme. The latter form stable complexes with the enzyme and are also inhibitors. 

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