Identification of Outer Mitochondrial Membrane Cytochrome b5 as a Modulator for Androgen Synthesis in Leydig Cells

Abstract

Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017α, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017α-catalyzed reactions, 17α-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17α-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17α-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells. Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017α, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017α-catalyzed reactions, 17α-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17α-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17α-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells. Two isoforms of cytochrome b5 are known to exist in a single cell. One is microsomal cytochrome b5 (b5) 1The abbreviations used are: b5, microsomal membrane cytochrome b5; OMb, outer mitochondrial membrane cytochrome b5; P-45017α, cytochrome P-45017α (cytochrome P-450 catalyzing the 17α-hydroxylation and C17-C20 bond cleavage of pregnenolone and progesterone); P-450scc, cytochrome P-450scc (cytochrome P-450 catalyzing the side chain cleavage of cholesterol); P-450 reductase, NADPH-cytochrome P-450 reductase; ER, endoplasmic reticulum; PBS, Na-Pi-buffered saline; StAR protein, steroidogenic acute regulatory protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. in the endoplasmic reticulum (ER), and the other is outer mitochondrial membrane cytochrome b5 (OMb) (1Fukushima K. Ito A. Omura T. Sato R. J. Biochem. (Tokyo). 1972; 71: 447-461PubMed Google Scholar, 2Ito A. J. Biochem. (Tokyo). 1980; 87: 63-71Crossref PubMed Scopus (62) Google Scholar, 3Ito A. J. Biochem. (Tokyo). 1980; 87: 73-80Crossref PubMed Scopus (23) Google Scholar). They consist of the following three domains: (a) the amino-terminal hydrophilic, (b) medial hydrophobic, and (c) carboxyl-terminal hydrophilic domains. A protoheme is bound to the first domain, which is highly conserved between b5 and OMb with 70% identity (4Lederer F. Ghrir R. Guiard B. Cortial S. Ito A. Eur. J. Biochem. 1983; 132: 95-102Crossref PubMed Scopus (89) Google Scholar, 5De Silvertis M. D'Arrigo A. Borgese N. FEBS Lett. 1995; 370: 69-74Crossref PubMed Scopus (50) Google Scholar). The hydrophobic domain, consisting of about 20 amino acid residues, is embedded in the lipid bilayer and functions for the insertion of proteins into the membranes as tail-anchored proteins (6Kutay U. Ahnert-Hilgen G. Hartmann E. Wiedenmann B. Rapoport T.A. EMBO J. 1995; 14: 217-223Crossref PubMed Scopus (263) Google Scholar). The carboxyl-terminal 10 amino acid residues of b5 are exposed to the luminal side of the ER cisterna (7Vergeres G. Ramsden J. Waskell L. J. Biol. Chem. 1995; 270: 3414-3422Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 8Kuroda R. Kinoshita J. Honsho M. Mitoma J. Ito A. J. Biochem. (Tokyo). 1996; 120: 828-833Crossref PubMed Scopus (31) Google Scholar) and are required for the targeting of the cytochrome to the organelle (9Mitoma J. Ito A. EMBO J. 1992; 11: 4197-4203Crossref PubMed Scopus (103) Google Scholar). The visible spectroscopic properties of the reduced forms are characteristic of the b5-type hemoproteins. OMb and b5 are spectrographically indistinguishable from each other due to the highly conserved heme-binding portion (1Fukushima K. Ito A. Omura T. Sato R. J. Biochem. (Tokyo). 1972; 71: 447-461PubMed Google Scholar, 2Ito A. J. Biochem. (Tokyo). 1980; 87: 63-71Crossref PubMed Scopus (62) Google Scholar). OMb and b5 have a similar mobility on SDS-PAGE and are co-purified unless specific precaution was taken in the purification procedures. Antibodies directed against b5 that had been purified without the removal step of OMb cross-react with OMb. In some case, cross-reactions are observed even if a highly purified form of OMb or b5 was immunized in rabbits. These facts made the discrimination between OMb and b5 difficult. The most convincing way of the discrimination is use of the specific antibodies. We have such antibodies in a large collection of anti-OMb and anti-b5 antibodies. Because b5 is a hemoprotein with sixth co-ordination, it is incapable of activating an oxygen molecule as cytochrome P-450 does. One of its physiological functions is an electron transfer to terminal oxidases such as stearyl-CoA desaturase (10Ozols J. Ann. Clin. Res. 1976; 8: 182-192PubMed Google Scholar). Some evidence also suggests that b5 functions as a modifier for some cytochrome P-450-catalyzed reactions although its mechanism is not clear (11Hilderbrandt A. Estabrook R.W. Arch. Biochem. Biophys. 1971; 143: 66-79Crossref PubMed Scopus (454) Google Scholar, 12Guengerich F.P. Martin M.V. Beaune P.H. Kremers P. Wolff T. Waxman D.J. J. Biol. Chem. 1986; 261: 5051-5060Abstract Full Text PDF PubMed Google Scholar). For example, b5 stimulates the 6β-hydroxylation of testosterone and nifedipine oxidation by recombinant CYP3A4 (13Yamazaki H. Useng Y.-F. Shimada T. Guengerich F.P. Biochemistry. 1995; 34: 8380-8383Crossref PubMed Scopus (140) Google Scholar). It also augments C17-C20 lyase activity of pig, guinea pig, and human P-45017α to of and and M. Biochem. Biophys. Res. PubMed Scopus Google Scholar, S. N. R. S. J. Biochem. Biol. 1992; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of b5, the lyase activity of the P-45017α is low to for physiological production of in The physiological functions of OMb are not The evidence for the is that specific antibodies against rat OMb reductase, which catalyzes of in outer mitochondrial membrane of rat cells A. S. T. Biochem. Biophys. Res. PubMed Scopus Google Scholar, H. Ito A. J. Biochem. (Tokyo). 1986; PubMed Scopus Google Scholar). In a on the mechanism for of OMb, we have observed that it from the outer mitochondrial membrane to the ER membrane in a of or to J. T. and A. in In other such as and of OMb was present in ER in to These that OMb the to localized in outer mitochondrial membranes or in ER although the of OMb to in the carboxyl-terminal 10 amino acid residues for mitochondrial targeting R. T. Honsho M. S. Mitoma J. Ito A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In guinea pig, OMb is in its is b5 is in rat OMb is between the outer mitochondrial membranes and ER membranes in high suggests that OMb and not b5 by the lyase activity of The indistinguishable properties between OMb and b5 as a that the effects on P-450 that have been to by b5 are by OMb. Thus, in we analyzed the effects of OMb on rat P-45017α, and we that cytochrome is a for in and guinea were from and were and in with the of and and of OMb and were with at a of and and testes were and using a in 10 and A. the were at for 10 the were at for 10 to the mitochondrial were by of the at for were at For of the of OMb and b5 in the the form the and were by H. G. Biochem. PubMed Scopus Google Scholar) and to The membrane was first with specific OMb or b5 and with and the were by using as were with and P-45017α and OMb were from rat testis microsomes with the 10 from and each of and and of the to to with 10 with the 10 the was with the 10 and The The was then with the 10 and with the 10 and The OMb. were purified by high on a with a of in the 10 P-45017α was to a and the was with the 10 without The was from the with the from and embedded with P-45017α were by a as S. S. A. S. Biophys. PubMed Scopus Google Scholar). using a OMb was also b5 was from microsomes and purified with the as OMb. NADPH-cytochrome P-450 reductase was from microsomes of and purified as S. T. S. and Scholar). P-45017α, OMb, and b5 were purified from the microsomal of the with the as the rat The OMb and b5 were from each other by the and of OMb and in E. amino-terminal OMb was by a The was into at the and to with was for at and then the was by at for the was at for The were in 10 and for at at for the was to a with 10 10 and with the the was with the Because effects of the recombinant OMb on the P-45017α-catalyzed were indistinguishable from those of the forms that were purified from testes and the recombinant OMb was used in of and C17-C20 of were with P-450 reductase and OMb or b5 in a of of and for at with 20 progesterone in was to the was with of at a concentration of and for at The was by of of and the were with the a of the were by high using a and with a acid and 17α-hydroxyprogesterone were by at In some 17α-hydroxyprogesterone was used as the and of P-45017α were using a of for the of the reduced form T. Sato R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of OMb and b5 were from the reduced between and by using a of of b5 that properties of forms of OMb and b5 were indistinguishable from that of a form of b5 at a T. Sato R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. S. J. Biochem. (Tokyo). PubMed Scopus Google Scholar). The concentration of P-450 reductase was from the at by using a of Arch. Biochem. Biophys. PubMed Scopus Google Scholar). of P-450 reductase in rat testis microsomes were analyzed by using P-450 and The of the was with and using were by a acid Biochem. PubMed Scopus Google Scholar). of OMb and were from and in with 10 at were then and embedded in were and using for In the were first with in Na-Pi-buffered for 20 at to the and then with of in for 20 at to the was by at with the first antibodies against rat OMb or guinea P-45017α that had been with rat for at The were then for at with to The proteins were with and as T. H. J. F. 1992; PubMed Scopus Google Scholar). was using For the first anti-OMb or was from The against P-45017α was a from S. of of OMb in antibodies used in are between OMb and anti-b5 between b5 and anti-OMb was observed if of the and of the antibodies were that OMb was between mitochondrial and microsomal at a of on a concentration in rat b5 was not A large amount of the on the SDS-PAGE the b5 which is that of cells. of OMb present in testes were with those in and of OMb was also observed in the and not of OMb in the cells had been to the outer mitochondrial of OMb from to the microsomal membranes was observed at of to Because not but also the amount of was in the microsomal of OMb to the microsomes was with other such as and not OMb a to in both the outer mitochondrial and microsomal In the of OMb as in the cells was not observed as by of or In to OMb, b5 not its in and other cells. and about the of OMb in cells are to J. T. and A. in of in the of the used in We purified P-45017α from rat testis microsomes and both b5 and P-450 reductase from rat We used a recombinant OMb form had the effects on rat P-45017α with the form that was purified from the testis of the recombinant form to the with a and of OMb. The were by using the recombinant OMb of the purified OMb. were purified from the the P-45017α, b5, and OMb were most in the The specific of P-45017α in rat testis microsomes was as from the of P-450scc, a mitochondrial protein, to the is for the microsomal as from with not The specific of P-450 reductase was from the of to OMb was present in the testis microsomes at an amount of as from the reduced The not in a microsomal with of b5-type of using excess of progesterone as the we both 17α-hydroxylase and lyase activities P-45017α first the to the and then the P-450-catalyzed the C17-C20 bond to without the a that from progesterone by guinea P-45017α and from pregnenolone by P-45017α H. S. S. Biochemistry. 1995; 34: PubMed Scopus Google Scholar, T. T. T. M. T. S. Biochemistry. PubMed Scopus Google Scholar). In the of of the intermediate from the in the and the bond cleavage with of the of both the and on of from P-450 reductase to P-45017α the cleavage is as in of was in a with the of P-450 reductase The amount of 17α-hydroxyprogesterone was in the These that the first step is and that the following the of the intermediate and bond are as H. S. S. Biochemistry. 1995; 34: PubMed Scopus Google Scholar). The of the electron transfer the P-450-catalyzed C17-C20 bond to without the of the The lyase activity was about the 17α-hydroxylase activity at a low of P-450 reductase to P-45017α of and it was the 17α-hydroxylase activity at a of P-450 reductase to P-45017α of rat P-45017α a lyase activity that is the 17α-hydroxylase activity with the guinea H. S. S. Biochemistry. 1995; 34: PubMed Scopus Google and K. S. S. Biophys. PubMed Scopus (62) Google and the of OMb and and of of OMb to the both the 17α-hydroxylase and lyase activities the of of P-450 reductase to P-45017α The to at a low of OMb to P-45017α the electron transfer was due to a low concentration of P-450 reductase the in Because the amount of the reductase is low in the testis microsomes as by with reductase we the effects of OMb at a of P-450 of unless OMb stimulated the 17α-hydroxylase activity at of OMb to P-45017α and to it to the of to an of of the lyase activity was also observed but a at a of OMb to P-45017α of about the of of the lyase activity was to the of whereas the 17α-hydroxylase activity to not of the lyase activity by OMb was about of b5 to the P-45017α and P-450 reductase also both lyase and 17α-hydroxylase activities but with a manner different that of OMb It stimulated the lyase activity to an similar to that of OMb at of b5 to P-45017α of whereas it the 17α-hydroxylase activity about at the At the high the 17α-hydroxylase and lyase activities to the and of the and the of not The in the effects between OMb and b5 were the activity was in the OMb had a to the whereas b5 stimulated the lyase A of the at a of b5 to of was due to the on the lyase in a high concentration of b5 to of OMb and and of P-45017α of most 17α-hydroxylase and lyase activities in the of b5 In the guinea P-45017α-catalyzed b5 been to the lyase activity without elevation of the 17α-hydroxylase activity K. S. S. Biophys. PubMed Scopus (62) Google Scholar). We purified b5 and OMb from microsomes of guinea and analyzed the effects of proteins the P-45017α that had also been purified from the membrane and embedded in b5 stimulated the lyase activity to a of b5 to guinea P-45017α of with of the it reduced both activities at the high The of rat b5 on guinea P-45017α was the on rat P-45017α that of the lyase was that of the 17α-hydroxylase and that the of both activities at of At a of rat b5 to guinea P-45017α of the in the lyase activity was about and that of the 17α-hydroxylase was b5 also stimulated the lyase activity to the of the activity was and the a The 17α-hydroxylase activity to to a of guinea b5 to guinea P-45017α of In the of rat OMb on guinea P-45017α was not observed the of the 17α-hydroxylase was observed in an manner to a of rat OMb to guinea P-45017α of was about The of guinea OMb on guinea P-45017α was intermediate between those of rat OMb and guinea b5 OMb stimulated the 17α-hydroxylase and lyase activities about and at a of of is a in the between the effects on guinea P-45017α by rat OMb and b5 of OMb and then analyzed the C17-C20 lyase activities 17α-hydroxyprogesterone using the as the b5 the lyase activity of rat P-45017α to a of b5 to P-45017α of about but the activity at the of a was the on guinea P-45017α by rat b5. OMb stimulated the lyase activity as the to rat P-45017α and the at the of in the with an excess amount of progesterone the of rat OMb to a on the lyase activity of guinea P-45017α 17α-hydroxyprogesterone was of OMb on P-45017α-catalyzed at a of on the effects of OMb and b5, which were were with a concentration of progesterone we of both 17α-hydroxylase activity and 17α-hydroxylase and C17-C20 lyase activities of P-45017α at the We then the P-45017α-catalyzed at a low concentration of such a a of the and at the first step 17α-hydroxylation of progesterone and of 17α-hydroxyprogesterone from the and at the step of the intermediate and its C17-C20 cleavage. At progesterone, rat a amount of with a 17α-hydroxyprogesterone In the of rat OMb progesterone to 17α-hydroxyprogesterone and to reflecting the an excess amount of progesterone as in The amount of 17α-hydroxyprogesterone was between and which the and were and then to and the intermediate was The of of progesterone and to have at the other production of and to at which most of the and intermediate were of rat b5 to the P-45017α-catalyzed stimulated of 17α-hydroxyprogesterone as as that of OMb the first and facilitated a of the intermediate It that of OMb at the first and the to and had by that of of and The for a amount of progesterone even of to an of on the P-45017α-catalyzed facilitated by b5 was that by OMb, of the at were the for the stimulated by OMb was that by P-45017α of rat OMb and b5 on guinea P-45017α were also in the reactions at a low concentration of b5 stimulated by guinea P-45017α in a similar to that by rat In rat OMb not effects on the production a of 17α-hydroxyprogesterone production not OMb in Leydig of cells are the following OMb present in the steroidogenic Leydig we using OMb on the rat OMb as as P-45017α was in Leydig reactions were observed in and In cells were not with b5 not of OMb in Leydig cells was also with the guinea testis not the of OMb have not been those of b5 are They are as follows: (a) transfer of from to such as desaturase (10Ozols J. Ann. Clin. Res. 1976; 8: 182-192PubMed Google Scholar) and S. J. Biol. Chem. Full Text PDF PubMed Google (b) of to K. Eur. J. Biochem. PubMed Scopus Google and (c) of some The of b5 is ER, whereas that of OMb is between outer mitochondrial and ER membranes from cells to cells or to OMb its at in cells different In Leydig cells of rat OMb is between outer mitochondrial and ER In b5, which is to for of the P-45017α of other in production of is in rat cells. In the present we analyzed the effects of rat OMb on P-45017α purified from rat testis microsomes using a with is to membrane proteins with a with the and to in such a way as the and from progesterone by guinea P-45017α H. S. S. Biochemistry. 1995; 34: PubMed Scopus Google Scholar) and from pregnenolone by P-45017α T. T. T. M. T. S. Biochemistry. PubMed Scopus Google Scholar) are to by a in the of excess of progesterone, in which electron from P-450 reductase to P-450 the C17-C20 cleavage of the bound to the The of OMb on rat reactions was also observed In the rat the amount of P-450 reductase, is from to of P-45017α We analyzed the effects of b5 and OMb at a of of that the physiological electron transfer between the reductase and P-450 in the rat testis ER at about different from the P-45017α of other the lyase activity is the 17α-hydroxylase activity in rat P-45017α if are it reduced the physiological concentration of P-450 In such a low concentration of P-450 reductase, production is due to the of the intermediate from the which the for the C17-C20 cleavage at an from a amount of progesterone by rat P-45017α in the of the physiological concentration of P-450 reductase is in the of b5 or OMb the of from progesterone is which is about of and not to to in the of an excess amount of progesterone in the T. T. T. M. T. S. Biochemistry. PubMed Scopus Google Scholar). b5 from the on the P-45017α by for the It elevated the lyase activity to a to that at a of P-450 P-450 of in the of b5. at a low of b5 to P-45017α and even to the at the high if the amount of P-450 reductase in the is The for such an of b5 is but to some of b5 with the reductase or even with P-45017α in a OMb also effects on rat P-45017α with some different The lyase activity was to the amount of OMb at low of OMb to P-45017α and a the of without a at the high in the 17α-hydroxylase activity was that in the lyase and the also to the of but to the of and of the for P-45017α between b5 and OMb was were for effects on guinea P-45017α, which a high 17α-hydroxylase activity to the lyase The effects of rat b5 were the for rat P-45017α, whereas rat OMb the 17α-hydroxylase activity without elevation of the lyase a of guinea P-45017α with rat b5 or rat OMb is it of the effects of similar In rat OMb is present in both the mitochondria and microsomes with a concentration with that in the rat OMb was localized in Leydig P-45017α In we b5 in the rat testis microsomes, not to its in the by using b5 not of b5 in Leydig cells. have been on of b5 on P-45017α rat M. Biochem. Biophys. Res. PubMed Scopus Google Scholar, S. N. R. S. J. Biochem. Biol. 1992; PubMed Scopus Google Scholar, J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Estabrook R.W. Biochemistry. PubMed Scopus Google Scholar). of the as a for the is on of OMb in the a on human OMb and P-45017α both in cells P. J. Biochem. Biol. PubMed Scopus Google Scholar). In the present and we were to the of OMb and not b5 as a physiological for rat rat OMb stimulates 17α-hydroxyprogesterone production from progesterone, it an activator for by In an excess amount of progesterone, rat P-45017α by the from the not physiological In steroidogenic the of is from a in the outer mitochondrial membrane to the membrane is present of steroidogenic acute regulatory 1996; PubMed Scopus Google Scholar). of StAR is by the of such as and The cholesterol is to pregnenolone by the and the pregnenolone is then by to a of The progesterone is to the in have step other then the side chain cleavage. a of by the with a low amount of progesterone as in the of At a low concentration of progesterone, OMb stimulates rat P-45017α to both 17α-hydroxyprogesterone and in which 17α-hydroxyprogesterone production is the 17α-hydroxyprogesterone and then as the but The intermediate is then to which is also stimulated by OMb. the of production stimulated by b5 is about as as that by OMb, the amount at was the for both hemoproteins. A then as to OMb and not b5 as the for production in the rat One the effects of b5 on P-45017α at a high of b5 In OMb not P-45017α activity at the of The by and M. Biochem. Biophys. Res. PubMed Scopus Google Scholar) of the effects between and testis b5 on production in testis The b5 stimulated the lyase whereas the b5 had an concentration for the OMb a to the 17α-hydroxylase activity of In rat P-45017α a 17α-hydroxylase activity to the P-45017α of other A high 17α-hydroxylase activity is for production by other which not due to the of P-45017α in the of the 17α-hydroxylase activity by rat OMb suggests that the high 17α-hydroxylase for an in Leydig cells. the of OMb in rat cells also have a physiological as a that between mitochondria and ER in Leydig although we have not analyzed of OMb in rat cells. are for the effects of b5 on some P-450-catalyzed One is that b5 is in the electron transfer the P-450 The in the first electron transfer is not the of the of P-450 is that of the of b5. For the electron transfer the of on testosterone 6β-hydroxylation and nifedipine oxidation by J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A is to P-450 between the P-450 and b5. Thus, the P-450 a stimulated activity in the The of P-45017α activities in the of b5 suggests some between proteins but with and The of the amount of P-450 reductase on the lyase activity of P-45017α in the present not in with the first The the are the the P-450-catalyzed and production of the of 17α-hydroxyprogesterone from the The effects of OMb in the preferential elevation of are not with the first the electron not the of T. T. T. M. T. S. Biochemistry. PubMed Scopus Google Scholar) that the of the intermediate the C17-C20 cleavage and the of the are to the first that 17α-hydroxylase and lyase activities are by the of the intermediate and the of the bond cleavage and the first step is the not even if the first step is by OMb or b5. Thus, at the of the for the of 17α-hydroxyprogesterone is by of the electron It is that rat OMb to the of 17α-hydroxyprogesterone and C17-C20 cleavage of androstenedione. is the first of the physiological of OMb. The functions in other as as the mechanism and of the between mitochondria and ER