Slow heme transfer from horseradish peroxidases C2 and A2, cytochrome c peroxidase, chloroperoxidase, and leghemoglobins to a heme acceptor protein, apomyoglobin, has been studied under mild conditions. The reaction is best described as heme release into water followed by quick engulfment by apomyoglobin. The energetics of the activated process are large and interpreted as connected to both polypeptide motions during release and the ordering of water around the heme during solvation. The free energy required to break the iron(III)-ligand 5 (L5) bond is a minor but crucial portion of the activation free energy. Donor-acceptor protein interactions are not involved in the transfer. Fast heme release from inactive protein has also been observed. Apoprotein recombination with porphyrins and hemes suggest that this lack of activity is a result of Fe-L5 bond breaking.The coenzyme and the prosthetic group share the role of being executive partners to a directing apoprotein. The former operates with an on/off relation to the protein during the functional cycle, whereas the latter remains attached. Heme, the archetypical prosthetic group, can, in a reversible but nonphysiological manner, be disconnected when the protein is perturbed by an acid, an alcohol, or a detergent (1). There is also a slow release of heme under mild conditions as regards temperature and ionic strength (1)(2)(3)(4) and at elevated temperatures (5). Release of heme from hemoglobin under mild conditions responded perceptibly to structural alterations due to single-point mutations (6). Some biological mechanisms seem to operate with a release of heme (7).Heme release from tryptophan 2,3-dioxygenase, a key enzyme in the regulation of tryptophan and serotonin, is critical for the synthesis and activity of this protein (8). In hemoglobinemias, the heme group may reside in variously modified pockets (9). A migration of heme between sites has been reported [e.g., MATERIALS AND METHODSHorseradish peroxidases A2 and C2 were isolated as described (14), and A2 was preliminarily fractioned on DEAEcellulose (pH 5.8-5.95), yielding subfractions that were homogeneous by discontinuous gel electrophoresis (15). Chloroperoxidase was purchased from Sigma. Lb a, c1, c2, d1, d2 were isolated from soybean nodules. apoMb has a higher affinity for Fe(III) hemes than other apoproteins, and heme release could be monitored by the formation of holoMb. The reaction was initiated by addition of a small (1%) volume of apoMb concentrate to the hemoprotein, usually in 50 mM phosphate/i mM EDTA, pH 7.0 (standard buffer). Heme transfer was monitored by the increase in holoMb absorbance at 409 nm or by the decrease in donor protein absorbance at <400 nm. Release was not observed from deoxyLb or carboxyLb.Data were analyzed by using either ENZFITTER (Biosoft) or RS/1 (BBN) programs. Kinetic parameters were calculated from the expressions §To whom reprint requests should be addressed. 882The publication costs of this article were defrayed in part by page charge payment. This a...

Ribonucleotide reductase is essential for DNA synthesis. In mammalian cells, the enzyme consists of two nonidentical subunits, proteins R1 and R2. The expression of the mouse R1 and R2 genes is strictly correlated to S phase. Using promoter-reporter gene constructs, we have defined a region of the TATA-less mouse ribonucleotide reductase R1 gene promoter that correlates reporter gene expression to S phase. This is demonstrated in stably transformed cells both synchronized by serum starvation and separated by centrifugal elutriation, suggesting that the R1 gene expression during the cell cycle is mainly regulated at the transcriptional level. The region contains four protein-binding DNA elements, ␤ (nucleotides ؊189 to ؊167), ␣ (؊98 to ؊76), Inr (؊4 to ؉16), and ␥ (؉34 to ؉61), together regulating promoter activity. The nearly identical upstream elements, ␣ and ␤, each form three DNA-protein complexes in gel shift assays. We have identified YY1 as a component in at least one of the complexes using supershift antibodies and a yeast one-hybrid screening of a mouse cDNA library using the ␣ element as a target. Transient transfection assays demonstrate that the ␣ and ␤ elements are mainly important for the R1 promoter strength and suggest that YY1 functions as an activator.The enzyme ribonucleotide reductase is essential for de novo synthesis of deoxyribonucleotides. In this perspective, ribonucleotide reductase plays a central role in providing the precursors for DNA synthesis. Mammalian ribonucleotide reductase consists of two non-identical homodimeric subunits, proteins R1 and R2 (1). Protein R1 (large subunit) contains the active site and allosteric sites for regulating enzyme activity. Protein R2 (small subunit) carries a tyrosyl free radical essential for enzyme activity. In quiescent or differentiated cells, none of the two subunits can be detected. In proliferating cells, the expression of protein R2 is S phase specific, whereas protein R1 shows a constant level throughout the cell cycle (2-4). However, both the R1 and R2 mRNA levels vary in parallel during the cell cycle with negligible levels during G 0 /G 1 and maximal levels during S phase (5, 6). The R1 and R2 promoters are also activated in resting cells upon UV irradiation leading to nucleotide excision repair (7).Genomic clones covering the mouse R1 and R2 genes have been isolated (8, 9). The R2 promoter contains a TATA-box and a proximal element, called ␣, that contains an NF-Y binding CCAAT motif (10).The R1 gene consists of 19 exons that covers 26 kb. 1 In contrast to the R2 promoter, the R1 promoter is TATA-less. DNase I footprinting assays proximal to the transcription start revealed two protected regions, ␣ (nt Ϫ98 to Ϫ76) and ␤ (nt Ϫ189 to Ϫ167) (8). These two regions are each 23 nt and identical except for one nucleotide. Three DNA-protein complexes, A, B, and C, are formed when oligonucleotides corresponding to the ␣ or ␤ footprints are used in gel shift assays. Complexes A and B are present at constant levels during the cell cycle, whereas c...

Mammalian ribonucleotide reductase (EC 1.17.4.1) is composed of two nonidentical subunits, proteins Rl and R2, both required for enzyme activity. The structure of the genomic mouse ribonucleotide reductase Ri gene was compiled from a number of overlapping A clones isolated from a Charon 4A mouse sperm genomic library. The Rl-encoding gene covers 26 kb and consists of 19 exons. All exon-intron boundaries were located by dideoxynucleotide sequencing, showing that intron 7 starts with the variant GC instead of GT. About 3.5 kb of DNA from the 5'-flanking region of the Rl-encoding gene were cloned and sequenced, and the transcriptional start site was determined by nuclease Si mapping of RNA. DNase I footprinting assays on the Ri promoter identified two nearly identical 23-bp-long protein-binding regions. Three protein complexes binding to one of the 23-mer regions were resolved and partially identified by using gel-retardation mobility-shift assays and UV crosslinking. One complex most likely contained Spl, and another complex showed S-phase-specific binding, suggesting a direct role in the cell-cycle-dependent Ri gene expression.Ribonucleotide reductase (EC 1.17.4.1) catalyzes the direct reduction of all four ribonucleotides to the corresponding deoxyribonucleotides, a reaction crucial for DNA synthesis (1, 2). Enzyme activity is strongly correlated to DNA synthesis, showing maximal activity in the S phase of the cell cycle.Mammalian ribonucleotide reductase is composed of two nonidentical homodimeric subunits, proteins Rl and R2, both required for activity. Each 45-kDa polypeptide of the R2 subunit contains a nonheme binuclear iron center that, during its formation, generates a tyrosyl-free radical essential for holoenzyme activity. The functional gene encoding the mouse R2 protein is mapped to chromosome 12 and has been cloned and sequenced (3, 4). The mammalian Rl subunit consists of two 90-kDa polypeptides containing the binding sites for nucleoside triphosphate allosteric effectors and ribonucleoside diphosphate substrates (5). The mouse Rl gene is mapped to chromosome 7, and full-length mouse Rl cDNA has been cloned and sequenced (6)(7)(8).The Rl protein can only be detected in proliferating cells, but the levels are constant and in excess throughout the cell cycle. Holoenzyme activity is regulated by an S-phasespecific de novo synthesis and subsequent breakdown of the R2 protein (9, 10). Both the Rl and R2 mRNA levels are undetectable in Go/Gl cells, rise dramatically and in parallel slightly preceding the entry of cells into the S phase, reach the same levels, and then decline as cells enter the G2 + M phases (11). The constant levels of the Rl protein through the different cell-cycle phases are most simply explained by its very long half-life compared to the R2 protein [Rl > 24 hr (12); R2 = 3 hr (9)].The S-phase-specific expression of R2 mRNA is regulated by an S-phase-specific release from a transcriptional blockThe publication costs of this article were defrayed in part by page charge payment. T...

The temperature dependence of the rates of heme release from the B subunits of methemoglobin A and 5 p mutant methemoglobins has been determined. The rates were largest for two hemoglobins with mutations distal to heme, previously known to be unstable. The other 3 mutants also released heme faster than A. These hemoglobins, with single point mutations at the cur& interface, were previously thought to be stable. The low reported yields of the 5 mutant proteins covaries with the relative rates of heme release from the met species.