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Rubredoxin-oxygen oxidoreductase (ROO) from the sulfate-reducing bacterium Desulfovibrio gigas, is an unusual terminal oxidase capable of reducing molecular O2 to water. The nature of its heme prosthetic groups has been investigated. It was found to contain two distinct hemes. One is Fe uroporphyrin I. The second is closely related to heme c. It is an Fe protoporphyrin IX where two electrophilic groups have added covalently across the double-bonds of the former 3,8 vinyl groups (IUB-IUPAC Tetrapyrrole numbering). These covalent attachment bonds are not to the main polypeptide chain, but instead are to an unidentified component. This component is soluble in acidic acetone, but cannot be extracted into organic solvents that typically solubilize free heme groups. Preliminary evidence suggests that the covalent bonds are not thioether bonds, but they are acid labile.

The biosynthetic origin of methyl groups in heme d1 isolated from the nitrite reductase cytochrome cd1 was investigated by a stable isotope labeling experiment. Pseudomonas aeruginosa (American Type Culture Collection strain 19429) was grown on a minimal medium supplemented with [13C]methionine. The enzyme was purified, the heme extracted, converted into the free base methyl ester derivative, and purified. 1H NMR and 13C NMR indicated that only the methyl groups attached to C2 and C7 are derived from methionine.

The substituents of the noncovalently associated heme prosthetic group of the bacterial nitrite reductase-cytochrome oxidase (EC 1.9.6.1 or EC 1.9.3.2.) from Pseudomonas aeruginosa (ATCC 19429) and Paracoccus denitrificans (ATCC 13456) have been identified. This was accomplished by 1H NMR, infrared, visible, and mass spectroscopic techniques applied to semi-purified heme and purified methyl ester derivatives of the iron-free porphyrin. The main structural features are as follows. 1) The macrocycle is a reduced porphyrin of the chlorin type, that is, one of the pyrrole rings has been saturated so that the beta-carbons have sp3-hybridization. 2) The chlorin is a tetracarboxylic acid. 3) The substituents of the saturated pyrrole ring are two methyl groups and two hydroxymethyl groups. 4) The substituents of the unsaturated pyrrole rings are: (i) two methyl groups (ii) a propionic acid (iii) an acrylic acid, and (iv) two directly bonded formic acid groups. No firm evidence has been obtained for the relative positions of these unsaturated beta-pyrrolic substituents around the macrocyclic ring, but a proposed structure will be discussed. Several previously inexplicable chemical properties of the heme in extracts or in the intact enzyme can be now interpreted in view of the proposed structure. The trivial name "acrylochlorin" is suggested for the macrocycle.

Nitrimyoglobin was formed in greater than 94% yield by a simple reaction between excess nitrite and horse heart metmyoglobin at pH 5.5. This dark green pigment was shown by 1H NMR spectroscopy to be a single, pure product with a well defined tertiary structure that is highly similar to the starting myoglobin. Electronic spin states parallel those of myoglobin, although the relaxation times differ. Ligand binding reactions of nitrimyoglobin parallel those of normal myoglobin, but lead to a unique series of UV-visible spectra. In the ferrous state, nitrimyoglobin reversibly binds O2 with half-saturation of sites at an O2 partial pressure of 10.4 +/- 1.4 mm Hg. 1H NMR data indicate that the altered heme of nitrimyoglobin has not undergone reaction at any meso proton position, nor has it been partially saturated to the level of a chlorin. 15N NMR spectra indicate that only a single nitrogen was added to the protein as a nitro group. Extraction of the modified heme from nitrimyoglobin and spectroscopic characterization of the nitriheme by infrared spectroscopy and of the free base porphyrin methyl ester derived from nitriheme by 1H NMR indicate that the modification is regiospecific. The heme in nitrimyoglobin is 3-(trans-2-nitrovinyl)-2,7,12,18-tetramethyl-8-vinylporphyrin-13,1 7-dipropionic acid. In the Fisher nomenclature scheme, the 2-vinyl substituent is the site of modification and has been converted to a nitrovinyl group by substitution of a proton by -NO2.

The amino acid sequence of Paracoccus (formerly Micrococcus) denitrificans cytochrome c550 has been established by a combination of standard chemical techniques and interpretation of a 2.5 A resolution x-ray electron density map. Peptides derived from a trypsin digest were chemically sequenced, and then ordered by fitting them to the density map. The amino acid compositions of chymotryptic peptides confirmed the x-ray map ordering the tryptic peptides. The amino acid sequence of this respiratory, prokaryotic cytochrome with 134 residues is discussed in relation to those of eukaryotic respiratory cytochrome c (103 to 113 amino acids), and prokaryotic, photosynthetic c2 (103 to 124 amino acids). At the primary structure level, c and c550 differ no more from cytochromes c2 than the various cytochromes c2 do from one another. It is suggested that the respiratory electron transport chain in prokaryotes and eukaryotes is a relatively late evolutionary offshoot of the photosynthetic electron transport chain in purple non-sulfur bacteria.

The configuration of the substituents around the macrocycle core has been determined for acrylochlorin, the reduced porphyrin that is the iron-free form of heme d1. This has been accomplished by two independent techniques. Nuclear Overhauser enhancements on the proton resonances of acrylochlorin d3-methyl ester were measured to determine nearest-neighbor substituents and mesoprotons. A lanthanide shift reagent, tris-(1,1,1,2,2,3,3-heptafluoro-7,7-dimethyloctane-4,6-di onato) europium(III), was found to bind in the vicinity of the carbonyl oxygens of the propionate and acrylate methyl esters. Differential chemical shift effects on the proton resonances of acrylochlorin provided evidence on relative distances from the lanthanide-binding site. Since the substituents are constrained along the circumference of the macrocycle, this provided information on configuration. Assuming that the dominant mechanism for spin-lattice relaxation in acrylochlorin in deuterated chloroform solution is proton dipole-dipole interaction, measured values of spin-lattice relaxation times could be interpreted as being consistent with the proposed configuration. The configuration bears sufficient similarity to that of protoporphyrin IX so that a common nomenclature may be used (Smith K. M. (ed) (1975) Porphyrins and Metalloporphyrins, pp. 3-5, Elsevier/North-Holland Biomedical Press, Amsterdam). The deduced configuration of the acrylochlorin methyl ester is: 1-methyl, 1-hydroxymethyl, 2-methyl, 2-hydroxymethyl, 3-methyl formate, 4-methyl formate, 5-methyl, 6-methyl propionate, 7-methyl acrylate, 8-methyl. The naturally occurring heme d1 has free carboxylic acids at the 3-, 4-, 6-, and 7-positions.

The stereospecificity of hydride transfer to NAD+ by several forms of rat liver aldehyde dehydrogenase was determined by a nuclear magnetic resonance method. The forms included several mitochondrial and microsomal isozymes from normal liver, as well as isozymes from xenobiotic-treated and tumor cells. The proton added to NAD+ comes exclusively from the aldehyde substrate and in all cases was A (pro-R)-stereospecific.