Residue Asn57 of bovine liver cytochrome b5 has been replaced with a cysteine residue, and the resulting variant has been isolated from recombinant Escherichia coli as a mixture of four major species: A, BI, BII, and C. A combination of electronic spectroscopy, 'H NMR spectroscopy, resonance Raman spectroscopy, electrospray mass spectrometry, and direct electrochemistry has been used to characterize these four major cytochrome derivatives. The red form A (Em = -19 mV) is found to possess a heme group bound covalently through a thioether linkage involving Cys57 and the a carbon of the heme 4-vinyl group. Form B, has a covalently bound heme group coupled through a thioether linkage involving the 13 carbon of the heme 4-vinyl group. Form BI, is similar to B, except that the sulfur involved in the thioether linkage is oxidized to a sulfoxide. The green form C (Em = 175 mV) possesses a noncovalently bound prosthetic group with spectroscopic properties characteristic of a chlorin. A mechanism is proposed for the generation of these derivatives, and the implications of these observations for the biosynthesis of cytochrome c and naturally occurring chlorin prosthetic groups are discussed.While iron-protoporphyrin IX is ubiquitous as a noncovalently bound prosthetic group in a wide variety of electrontransfer proteins and enzymes, other metalloporphyrins that vary in the type and degree of substitution in the macrocycle are known to be important in biological systems (1). These include hemes a, c, o, and d (2), the last of which represents the general class of metallochlorins-i.e., metalloporphyrins in which one pyrrole ring is reduced.Site-directed mutagenesis is an established method for generation of metalloprotein variants as a means of gaining mechanistic insight into electron-transfer processes (3). We now report a cytochrome b5 variant that is representative of a class of heme protein mutants in which the native prosthetic group undergoes specific chemical modifications. As these modifications are related to the classical designations ofheme proteins, we refer to this phenomenon in which transformation between heme types results from site-specific protein mutation as transmutation. Characterization of the present cytochrome b5 variant and its properties, combined with similar studies of subsequent variants based on related principles, promises to provide useful insight into the mechanisms involved in the covalent attachment of the heme prosthetic group to cytochromes c and in chlorin biosynthesis. The principles to be learned from this variant should lead to the design of metalloproteins with specifically modified prosthetic groups possessing useful enzymatic and/or electron-transfer properties.

Halorhodopsin of Natronobacterium pharaonis which acts as a light-driven chloride pump is studied by time-resolved resonance Raman spectroscopy. In single-beam experiments, resonance Raman spectra were obtained of the parent state HR578 and the first thermal intermediate HR520. The parent state is structural heterogeneous including ca. 80% all-trans and 20% 13-cis isomers. The resonance Raman spectra indicate that the all-trans conformer exhibits essentially the same chromophoric structure as in the parent states of bacteriorhodopsin or halorhodopsin from Halobacterium salinarium. Special emphasis of the resonance Raman spectroscopic analysis was laid on the C=C and C=N stretching region in order to probe the interactions between the protonated Schiff base and various bound anions (chloride, bromide, iodide). These investigations were paralleled by spectroscopic studies of retinal Schiff base model complexes in different solvents in an attempt to determine the various parameters which control the C=C and C=N stretching frequencies. From these data, it was concluded that in the parent state the anion is not involved in hydrogen bonding interactions with the Schiff base proton but is presumably bound to a nearby (positively charged) amino acid residue. On the other hand, the anion still exerts an appreciable effect on the chromophore structure which is, for instance, reflected by the variation of the isomer composition in the presence of different anions and in the anion-depleted form. In contrast to the parent state, the intermediate HR520 reveals frequency shifts of the C=N stretching in the presence of different anions. These findings indicate a closer proximity of the bound anion to the Schiff base proton which is sufficient for hydrogen bonding interactions. These changes of the anion-chromophore interaction upon transition from HR578 to HR520 may be related to the coupling of the chromophore movement with the anion translocation.

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