JOURNAL OF BACTERIOLOGY, Dec. 1979, p. 987-995 Vol. 140, No. 3 0021-9193/79/12-0987/09$02.00/0 Manganese, an Essential Trace Element for N2 Fixation By Rhodospirillum rubrum and Rhodopseudomonas capsulata: Role in Regulation DUANE C. YOCH Department ofBiology, University ofSouth Carolina, Columbia, South Carolina 29208 Received for publication 3 August 1979 Nitrogenase (N2ase) from the photosynthetic bacterium Rhodospirillum rub- rum can exist in two forms, an unregulated form (N2ase A) and a regulatory form (N2ase R), the latter being identified in vitro by its need for activation by a Mn2+_ dependent N2ase activating system. The physiological significance of this Mn2+- dependent N2ase activating system was suggested here by observations that growth of R. rubrum and Rhodopseudomonas capsulata on N2 gas (a condition that produces active N2ase R) required Mn2+, but growth on or gluta- mate did not. Manganese could not be shown to be required for the biosynthesis of either nitrogenase or synthetase or for glutamine synthetase turn- over, but it was required for the in vitro activation of N2ases from N2 and glutamate-grown R. rubrum and R. capsulata cells. Chromatium N2ase, in contrast, was always fully active and did not require Mn2+ activation, suggesting that only the purple nonsulfur are capable of controlling their N2ase activity by this new type of regulatory system. Although R. rubrum could not substitute Fe2+ for Mn2+ in the in vivo N2 fixation process, Fe2+ and, to a lesser extent, Co2, could substitute for Mn2+ in the in vitro activation of N2ase. Electron paramagnetic resonance spectroscopy of buffer-washed R. rubrum chromato- phores showed lines characteristic of Mn2+. Removal of the Mn2+-dependent N2ase activating factor by a salt wash of the chromatophores removed 90% of the Mn2 , which suggested a specific coupling of this metal to the activating factor. The data presented here all indicate that Mn2+ plays an important physiological role in regulating the N2 fixation process by these photosynthetic bacteria. A role for manganese (Mn2") in the Both forms of N2ase are interconvertable in vivo fixation process was implicated several years ago under a variety of conditions related to the cells when it was shown that Mn2+ greatly enhanced available nitrogen substrate (or lack of it) (5). nitrogenase (N2ase) activity in extracts from the The relationship between these two forms of photosynthetic bacterium Rhodospirillum rub- N2ase is shown by equation 1: rum (17, 24). Manganese was shown in these studies to act in conjunction with a chromato- Growth on N2 phore-bound or glutamate "activating factor" and ATP to I N2ase A - N2ase R (1) activate the Fe protein of N2ase in extracts pre- N starvation pared from glutamate (18) or N2-grown cells (25). Once activated, the Fe protein could func- Although N2ase A and R appear to be an integral tion normally with MoFe protein to form an part of a system which regulates N2ase activity active N2ase complex (17, 18,24), and Mn2+ was in R. rubrum, a link has not, however, been not required for catalysis. It was subsequently established between N2ase regulation in vivo shown (5) that R. rubrum N2ase could exist in and the in vitro N2ase R activation process (17) two chemically defined forms. One form, N2ase represented by equation 2. A, found only in nitrogen-starved celLs, was fully Activating factor active without the activating cofactors, whereas Mn2+, ATP a second form of the enzyme, whose activity is N2ase Rinactive N2ase Ractive (2) capable of being regulated, was isolated from Spontaneous during N2- or glutamate-grown cells and required a cell disruption Mn2' activating factor system for its activation; For reasons not yet understood, N2ase is always this form of the enzyme was called N2ase R. inactive in extracts prepared from either N2- or 987 988 YOCH J. BBACTERIOL. glutamate-grown cells and must be activated cated, all cells used for the various enzyme prepara- before its activity can be measured. Because tions were cultured in media containing the regular both inactivation of N2ase and its reactivation concentrations of manganese. by the Mn2+-dependent process are observed All glassware used in studies to determine Mn2" only in vitro, it seemed necessary to demonstrate requirements was rinsed four or five times with double- distilled water. Growth media for these studies were that this reaction (equation 2) was also func- also prepared with double-distilled water. These pre- tional at the cellular level if, as we believe, the cautions seemed to be adequate for keeping most activity of N2ase R is really modulated by the contaminating Mn2" out of the media because after cell in response to changes in its nutritional several passages in minus-Mn2" media, N2-fixing cul- environment. Since N2ase R must be active in tures could be shown to have a requirement for this vivo when cells are growing on N2, it seemed trace element. likely that if the Mn2+-dependent N2ase activat- Cultures (except those fixing N2) were grown in 1-, ing system was of physiological significance, it 2-, or 4-liter glass-stoppered Pyrex bottles filled com- might be possible to show an Mn2" requirement pletely to exclude air. Nitrogen-fixing cultures were grown in 250-ml side-arm flasks containing 50 ml of for R. rubrum growing on N2. media. After inoculation, these flask were evacuated This communication describes experiments and refilled at least four times with N2 gas. Illumina- showing that indeed Mn2" is required for growth tion was provided by placing the cultures between two on N2 and that this requirement cannot be ex- banks of four 40-W fluorescent lights (Vita-Lite, Duro- plained by any process related to nitrogen fixa- Test Corp., North Bergen, N.J.). This system provided tion except the activation of N2ase R. N2ase A about 950 footcandles of light at the surface of the and R were also found in Rhodopseudomonas culture bottles. Culture temperatures were maintained capsulata, thus extending this unique N2ase reg- between 25 and 300C. to a second of Extract preparations. All N2ase extracts were ulatory system species photosyn- prepared from freshly harvested cells which were re- thetic bacteria. This regulatory system may, suspended in approximately 2 volumes of argon-satu- however, be limited to the family Rhodospiril- rated 330 mM tricine buffer (pH 8.5) containing 4 mM laceae, since Chromatium vinosum N2ase did sodium dithionite. The resuspended cells before being not respond under any condition to Mn2' and broken were placed in a flask which was closed with a activating factor. serum bottle stopper and evacuated and flushed with argon several times. Cells were disrupted by sonic MATERLALS AND METHODS oscillation with a Sonifier cell disruptor (Heat Systems Inc.) at 65 W output for two 15-s periods. The soni- Bacterial strains. Both R. rubrum strain S-1 and cation vessel was a 60-rnl glass beaker covered with C. vinosum (fornerly Chromatium D) were single- Parafilm with a hole in it just large enough to insert colony (N2-fixing) isolates of the parent strains ob- the sonicator probe. After the probe was inserted, this tained from D. I. Arnon. R. capsulata strain B-10 was closed vessel was purged with argon 5 min before the kindly provided by J. D. Wall (initially from B. Marrs). cell suspension was transferred to it anaerobically by Media and bacterial growth conditions. R. rub- syringe; the gas flow through the vessel was main- rum and R. capsulata were grown photosynthetically tained during the sonication period. The broken cells on the medium of Ormerod et al. (26) supplemented were transferred by syringe to a capped, degassed with 20 mM malate. (For growth of R. capsulata, centrifuge tube and centrifuged at 30,000 x g for 10 thiamine [300 ug/liter] replaced biotin.) The nitrogen min. The pellet was discarded, and the supernatant source for this medium (26) was modified for the extract was either used directly as a source of N2ase various experiments as follows. (i) In experiments to and activating factor or was further fractionated by test the cells' ability to grow in the absence of Mn2" centrifugation at 240,000 x g for 90 min. The super- (Fig. 1 and 2), the nitrogen source was either N2 gas, natant fraction from the high-speed centrifugation 20 mM glutamate, or 10 mM (NH4)2SO4, as indicated. contained the soluble N2ase, and the pellet contained (ii) For testing the cells' ability to derepress N2ase in the chromatophore membranes (to which the N2ase the absence of Mn2+ (Fig. 3) and for cells containing activating factor was bound). high levels of glutamine synthetase (Fig. 4), growth- Enzyme assays. N2ase was assayed and ethylene limiting concentrations of NH4' (2.5 mM) were used was determined as described by Carithers et al. (5). to ensure eventual nitrogen starvation of the culture. Biosynthetic glutamine synthetase activity was as- (iii) To obtain cells which contained predominately sayed as described by Shapiro and Stadtman (29); the N2ase R, the nitrogen source was either growth-limit- procedure of Bender et al. (2) was used for the y- ing concentrations of NH4' (2.5 mM) followed by the glutamyl transferase assay of this enzyme. The gluta- addition of 0.75 mM glutamate after the N2ase was mine synthetase adenylylation number (ii) was deter- completely derepressed (Fig. 4) or, alternatively, grow- mined as previously described (30). Reaction mixture ing the cells on 5 mM glutamate to the point of constituents for these assays are found in the appro- nitrogen starvation (as evidenced by the vigorous pho- priate figure legends. toevolution of H2). C. vinosum was grown photosyn- Protein was determined by the biuret procedure thetically on a medium described by Arnon et al. (1) (10) with bovine serum albumin as the standard. Man- which was modified by replacing the NH4Cl with 20 ganese was determined on a Perkin-Elmer (model 503) mM glutamate as the nitrogen source. Unless indi- flame ionization spectrometer. VOL. 140, 1979 ROLE OF MANGANESE IN N2ase REGULATION 989 RESULTS thus does not require Mn2" for activation. Iso- lates from these cultures are currently under Effect of manganese on growth. Nitrogen- investigation. fixing organisms have long been known to re- R. capsulata showed an almost identical re- quire molybdenum (3) and higher than usual sponse as R. rubrum for Mn2" when growing on amounts of iron (12); more recently magnesium N2 (Fig. 2), suggesting that this member of the was shown to be required for N2ase activity in family might also produce an vitro (7). The observation that Mn2' was re- Mn2+-requiring form of N2ase when grown on quired for activation ofthe R. rubrum Fe protein N2. Although not as many growth experiments (17, 24) and the suggestion that this phenome- were carried out as with R. rubrum, the same non was involved in the regulation ofthe enzyme general pattern of response to Mn2+ was ob- (5, 18) prompted an investigation to determine served, in that Mn2+ was required only for whether Mn + was required for growth of this growth on N2 and not when glutamate or am- organism. The results of numerous growth ex- monia were the nitrogen sources (Fig. 2). periments indicate that only when R. rubrum Although both N2- and glutamate-grown cells was grown on N2 gas could an absolute require- produce the Mn2+-dependent species of N2ase ment for Mn2' be shown (Fig. 1A). In several of (N2ase R) (5), only those cells forced to use this the experiments, however, (out of a total of 16) enzyme (those required to grow on N2) in fact a lag period of 60 to 100 h was observed in required the addition of Mn2+ to the growth minus-Mn2+ media which was followed by media. It must be pointed out that the Mn2+- growth at near nornal rates. In contrast, no free media containing either glutamate or NH4' difference in either the rate or extent of growth actually contained about 0.15 ,uM Mn2+ (com- of R. rubrum was ever observed in the absence pared with 15 ,uM in plus-Mn2' media), and this of Mn2' with either glutamate or ammonia as level may have been sufficient for the cells' more the nitrogen source (Fig. 1B). The growth of R. limited needs for growth on these nitrogen rubrum on N2 (minus Mn2+) which followed the sources. The observation that cells required long lag period suggests that a mutant may arise Mn2+ for growth on N2 only after they had been whose N2ase R is locked into an active state and carried through two or three passages of Mn2+-

500 A. 400 300

200 N2 E 2. c A plus MN 0 ID A 2 ¶@100 minus MN2 4# 0io

z so

40

HOURS FIG. 1. Effect of Mn2" on the growth of R. rubrum with either N2, glutamate, or NH4' as the nitrogen source. Inocula for these growth experiments were obtained from N2-fixing cells that had been cultured for at least onepassage in Mn2.-free growth media. Cells were grown in static culture (plus or minus Mn24) with the nitrogen source as indicated (1 atm ofN2 or 10 mM of either glutamate or NH42). 990 YOCH J. BACTERIOL. N2

200 sunus M-n

(aso z

200

20 A minus MN2+

A ^ 10 HOURS

I I I I I I I p I FIG. 2. Effect of Mn2+ on the growth of R. capsulata on different nitrogen substrates. Conditions were identical to those in Fig. 1. deficient media suggests that they are capable of Mn2+-deficient media to insure low levels of of storing variable amounts of this metal. This the element in the cell. Not only did N2ase is confirmed by the large Mn2" electron para- synthesis proceed without Mn2+ (in vitro analy- magnetic resonance signal observed from iso- sis showed it to be N2ase A), but high levels of lated chromatophores (see figure below). Be- glutamine synthesis activity were also found in cause of this storage capacity and the low Mn2" these cells (unpublished data), indicating that concentrations contaminating the "Mn2+-free" the Mn2" requirement for N2-flxing cultures was media, a quantitative determination of the not required for the synthesis of either of these amount of Mn2+ required for cell growth on N2 two critical enzymes. was not attempted. Since glutamine synthetase activation is Is Mn2+ required for N2ase synthesis or known to require either Mg2e or Mn2", with the glutamine synthetase activity? Growth on specific element depending on the organism (6, N2 gas requires that an organism synthesize both 13, 14, 30), the biosynthesis of glutamine by nitrogenase and glutamine synthetase, the latter glutamine synthetase was examined for its metal being needed to metabolize the newly formed requirement. The enzyme from both R. rubrum NH4' (22, 31). Because glutamate synthase (also and R. capsulata nitrogen-starved cultures was required by N2-fixing cells) is found in significant specific for Mg2e and not Mn2' (Fig. 4). The concentrations in NH4+-grown R. rubrum cells greater relative effectiveness of Mn2+ with the (31) and cells presumably containing this en- R. capsulata enzyme was probably a result of zyme grew well on NH4+ without Mn2+, it is its greater degree of adenylylation (ni = 5) com- assumed that the Mn2' dependence for growth pared to that of R. rubrum (ni = 3), a factor on N2 (Fig. 1A) is not due to a requirement by known to have some influence on the metal ion glutamate synthase. To determine whether the specificity of the biosynthetic reaction (15, 16). need for Mn2' by R. rubrum growing on N2 It should also be noted that the glutamine syn- might be required for N2ase synthesis, the rate thetase from R. capsulata required a much and level of N2ase activity were measured in a lower cation concentration for optimal biosyn- culture grown on NH4+-limiting media where thetic activity than did the enzyme from R. N2ase synthesis would normally follow exhaus- rubrum, suggesting an interesting, but at the tion of the nitrogen supply. As shown in Fig. 3, moment unexplainable, difference between the derepression of N2ase proceeded identically in enzymes from these two organisms. media both with and without added Mn2+. To Once a Mn2"deficient culture had been ob- insure that intracellular Mn2+ was not support- tained, it could be determined whether this ele- ing the synthesis of N2ase, the inoculum used ment was involved in the conversion by the cell here had previously been through five passages of N2ase A to the R form. To answer this ques- VOL. 140, 1979 ROLE OF MANGANESE IN N2ase REGULATION 991 The Mn2" requirement of N2 fixation by R. 20 capsulata cultures (Fig. 2) suggested that this purple nonsulfur bacterium regulates its N2ase in a manner similar to R. rubrum. This hypoth- E 15 esis was tested by growing R. capsulata under C.-7 conditions which are known in R. rubrum to produce either N2ase R (growth on 5 mM glu- z EL' /plus MN"2 tamate) or N2ase A (nitrogen starvation) and LLI ~~~~~minus MN2* testing the N2ases from these cultures for their 00C. need for Mn2' and activating factor. Figure 5B ~.e 5 shows R. capsulata N2ases to have a kinetic pattern identical to that of R. rubrum (Fig. 5A), that is, N2ase from glutamate-grown cells showed a lag period and required Mn2+ for max- 0 10 20 30 imum activity, a characteristic of N2ase R. Con- HOURS trols (not shown here) indicated that the acti- was also for this FIG. 3. Effect of Mn2+ on the synthesis of R. rub- vating factor protein necessary rum N2ase. R. rubrum was grown on 1.25 mM Mn2+-enhanced N2ase activity. Alternatively, (NH4)2SO4, a concentration that assures nitrogen N2ase activity from N-starved R. capsulata cells starvation in the mid-logphase ofgrowth. Inoculum was linear from zero time in the absence of Mn2+ for the R. rubrum culture that was to be tested for (Fig. 5B), which is indicative of N2ase A. These derepression in the absence ofMn24 was taken from data demonstrate unequivocally that R. capsu- a culture that had been through four passages in lata, like R. rubrum, produces two forms of media without Mn2+ to insure that the cell's internal N2ase, types A and R, and that the activity of concentration of this element had been depleted. N2ase R is further regulated by a chromato- Each point on the graph represents the activity of) ml ofculture in a 7-ml Fernback flask evacuated and phore-bound Mn24-dependent activating factor. flushed several times with high-purity argon. The A similar analysis of C. vinosum N2ase showed cells were preincubated in the light (I = 2 x 104 ergs that it was fully active when grown on either per cm2 per s) 15 min before the addition ofacetylene glutamate (Fig. 5C) or N2 (unpublished data) (4%) to the flask. The cells were then incubated in the presence of acetylene for 20 min, at which time they were killed by the addition of 0.1 ml of 10% trichlo- roacetic acid; the ethylene produced was determined by gas chromatography. tion, Mn2+-deficient cultures ofR. rubrum which had been starved for nitrogen (a condition under which they produce N2ase A) were fed sodium glutamate, which is one of several treatments which normally causes the conversion of N2ase A to the R forn (5). It was found that Mn2+- deficiency did not impair the conversion ofN2ase A to the R form. Effect of MnA" on N2ase activity. Since the requirement for Mn24 by N2-fixing cultures of R. [ Cation ] mM rubrum and R. capsulata could not be explained FIG. 4. Cation specificities of R. rubrum and R. by a role in either protein synthesis, glutamine capsulata glutamine synthetase (biosynthetic activ- synthetase turnover, or the conversion of N2ase ity). The reaction mixture contained 50 mM imidaz- A to R, one is left with the possibility that this ole-hydrochloride buffer (pH 7.45), 100 mM sodium trace element is required for the regulation of glutamate, 50 mM NH4Cl, 3.0 mM ATP, and either N2ase activity. The influence of Mn2+ on the R. rubrum (180 pg ofprotein) or R. capsulata (300 pg activity of R. rubrum N2ase R in cell extracts is ofprotein) cell extract (supernatant from centrifuga- seen in Fig. 5A and is consistent with previous tion at 240,000 x g for 90 min). The concentrations of Mg2+ and Mn2+ were varied as indicated. The reac- observations (5, 17, 24). The nonlinear kinetics tion period was 10 min; the temperature was 37°C. of the reaction are indicative of an activation The adenylylation number (ni) of the R. rubrum and process and, taken with the growth data (Fig. 1 R. capsulata enzymes was determined by the y-glu- and 2), suggest that the Mn24-dependent N2ase tamyltransferase reaction in the presence and ab- activation system plays an essential role in the sence of 60 mM Mg2+ and was calculated to be 3.4 N2 fixation process at the cell level. and 5.0, respectively. 992 YOCH J. BACTERIOL.

1600 2 1200 1200 plus MN Plus MN2- plus MN2

1250 1000 1000

XE 1000 / Boo

minus MN 0Z6) 750 600 S00

500 minus MN2 40 minus M2 400 S

260 0 200 200 0

0 10 20 30 0 10 20 30 0 10 20 30 MINUTES FIG. 5. Manganese requirement for the of photosynthetic bacteria. The complete reaction mixture contained in a final volume of 1.5 ml: 50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, (pH, 74), 25 mMphosphocreatine, 20 pg of creatine phosphokinase, 15 mM MgCl2, 2.9 mM ATP, 33 mM dithionite (8 mM dithionite used with R. capsulata nitrogenase), and activating factor (supplied by the addition of chromatophores (0.08 mg of ) to the reaction mixture. Cell extracts (supernatants from centrifugation at 240,000 x g for 90 min) containing N2ase R prepared from glutamate- grown cells (see text) were added at the following concentrations: R. rubrum, 2.9 mg ofprotein; R. capsulata, 3.1 mg ofprotein; and C. vinosum, 2.4 mg ofprotein. (R. capsulata N2ase A prepared from N-starved cells was added to the reaction mixture at a concentration of 2.9 mg ofprotein) N2ase from glutamate-grown cells assayed in thepresence of0.5 mM Mn2+ (@) and in its absence (0); N2ase from N-starved cells assayed in the absence ofMn2+ (A). Gasphase, 96% argon-4% acetylene. The vessels were shaken in a 30°C water bath, and gas samples were taken for the gas chromatographic analysis of ethylene at the times indicated. and did not respond to either Mn2" or Mn2" plus other divalent cations tested, only Co2+ was ef- activating factor. These results are in agreement fective, but only at very high concentrations with earlier observations that C. vinosum N2ase (Fig. 6, insert, and Table 1). Cobalt, however, activity was immediately linear with time (33) was active in this system only when the N2ase and did not require activation cofactors (9, 32, was derived from cells cultured in Mn2+-deficient 33). media, which may explain why Nordlund et al. Effectiveness of various divalent cations (25) found this element ineffective in stimulating in activating N2ase R. It has been suggested R. rubrum N2ase activity. that Fe2+ is equally as effective as Mn2" in the If one compares the properties of the divalent activation of R. rubrum N2ase in vitro (25). The cations that interact with the activating factor, failure of R. rubrum and R. capsulata, however, it appears that the order of efficiency, Mn2+ > to fix N2 in a growth medium deficient in Mn2+ Fe2+ > Co2+ > Zn2+, is related in some way to but rich in iron (0.5 mg/ml suggests that this the ionic radius of the metal, with those closest element cannot substitute for Mn2+ in the cel- to 0.079 nm in radius being the most effective lular regulation of N2ase. An examination of a (see Table 1). There also seems to be a correla- number of cations for their ability to function tion between ability to activate Fe protein and with the activating factor in the activation of the configuration of the metal complex, that is, N2ase R in vitro is shown in Fig. 6. Although only those metals which can exist in the octa- Fe2" substitutes for Mn2' anid the maximal ve- hedral configuration are recognized by the acti- locities (V) obtained with these two cations were vating factor. comparable, it is not nearly as effective as Mn2+ Evidence for a Mn2+-activating factor on a mole basis. Crude extracts, with activating complex on the chromatophore membrane. factor still bound to the chromatophore mem- Additional evidence which points toward a phys- branes, were used in these experiments to sim- iological role for the Mn2+-dependent activating ulate as closely as possible the in vivo situation; factor in regulating R. rubrum N2ase is the fact however, similar data were obtained using a that Mn2+ is bound to the chromatophore mem- semipurified, reconstituted system. Ofnumerous brane which is the intracellular site of the acti- VOL. 140, 1979 ROLE OF MANGANESE IN N2ase REGULATION 993 per mg of bacteriochlorophyll, whereas mem- branes washed with 0.5 M NaCl (a process which removes the activating factor from the mem- brane) had approximately 0.15 ,g of Mn2+ per

~~~~~~~~~~~~o2' mg of bacteriochlorophyll or about an order of magnitude less Mn2+ when the activating factor so~~ ~ ~~~~c had been removed. '~ C,) %.- z; Ev#402 0 DISCUSSION One of the key questions in the study of nitro- 0~~~~~~~2 gen fixation the past few years has concerned its mode of regulation, with most of this attention being focused on factors controlling its synthesis (21). We have previously found (5) that, in ad- dition to the normal repression-derepression sys- tem, R. rubrum can also regulate N2ase by con- trolling its level of activity. It does this by con- 0 0.2 0.4 0.6 0.8 1.0 verting an active unregulated form of the en- zyme (N2ase A) to a form (N2ase R) whose [Cation], mM activity can be regulated by a Mn2+-dependent FIG. 6. Divalent cation specificity of the N2ase R activating factor (5). The conversion of N2ase to activation system. Activating factor activity was the regulatory form occurs in response to measured by its ability to convert inactive N2ase R to changes in the nitrogen substrate of the cell and the active form. The reaction mixture was the same thus provides an explanation at the molecular as that described for Fig. 5, except that 1.4 mg of cell level for the observation that NH4' and gluta- extract (supernatant from centrifugateon at 30,000 x mine immediately inhibit N2ase activity in whole g) was used as both the source of N2ase R and cells (23, 28). This mechanism gives R. rubrum activating factor. Extracts containing N2ase R were the to prepared from cultures fully developed on 5 mM ability respond immediately to changes in glutamate. Divalent cations were added at the con- centrations indicated. N2ase activities were assayed TABLE 1. Specificity of N2ase R activating factor after 20 min ofincubation, and the rates expressed in for divalent cations and some physical the figure are averages of this period. characteristics ofthese ions Nonhy- vating factor (17). The Mn2+ is bound in that it Cation Va Km(mM) drated Favored configura- cannot be removed from the membrane by a ionic tion of complex' radius" neutral buffer wash. Electron paramagnetic res- onance spectra of washed chromatophores show Mg2+ 0C 0.66 Tetrahedral the characteristic six lines centered near 3,200 G Ca2+ 0 0.99 which result from hyperfine splitting ofthe Mn2+ Zn2+ 0 0.73 Tetrahedral X-band c02+ 13.3 1.62 0.74 Octahedral, tetra- signal (Fig. 7). The membrane-bound hedral, pyrami- Mn2" signal located to the left of the large bac- dal teriochlorophyll free radical signal at g-2.0 Ni2+ 0 0.71 Square, tetrahe- closely resembles that produced by purified dral, pyramidal Mn2+-concanavalin A (27) and is almost identi- Fe2+ 45 0.50 0.75 Octahedral cal to Mn(H2O)62+ (cf. Fig. 1, reference 27). The Mn2+ 35 0.19 0.79 Octahedral Mn2+ signal is prominent in both the isolated Cu2+ 0 0.72 Square (no addition) and dithionite-reduced mem- a Maximal velocity, nanomoles of C2H4 formed per branes; the latter treatment reduces the mem- min by activated N2ase R; in these experiments, the brane-bound succinate dehydrogenase which ac- initial velocity (V) was determined by averaging the counts for the signals at g-2.03, 1.93, and 1.91 (4), activity over a 20-min incubation period. but has no effect on the Mn2+ signal. b From Malmstrom and Rosenberg (20). Preliminary evidence strongly suggests that c N2ase catalysis requires Mg2+ (as Mg-ATP); there- the Mn2+ is actually associated with the activat- fore, all reaction mixtures contain this cation at a ing factor. This evidence comes from atomic concentration of 15 mM. Although a low rate of N2ase absorption spectroscopy activity in the absence of an additional cation is seen analysis, which showed in Fig. 6, this activity is due to a contaminating N2ase that buffer-washed membranes (whose electron A as previous studies (reference 4) have shown that paramagnetic resonance spectrum is shown in purified N2ase R is completely inactive without the Fig. 7) contained approximately 1.5 jig of Mn2+ activating system. 994 YOCH J. BACTERIOL.

Magnetic field (gauss) FIG. 7. Low-temperature electron paramagnetic resonance spectra of R. rubrum chromatophores. Chro- matophores from R. rubrum (1.0 mg of bacteriochlorophyllper ml) were suspended in 50 mM Tris-hydrochlo- ride buffer (pH 8.0). Although this particular batch of chromatophores was from NH4+-grown cells, these membranes have activating factor bound to them (17, 18; Chan and Yoch, unpublished observations). Top curve, electron paramagnetic resonance spectrum of buffer washed chromatophores. Bottom curve, electron paramagnetic resonance spectrum after addition ofa small quantity ofsolid sodium dithionite. Temperature, 35°K; power to the cavity, 5 mW; field modulation, 8.0 G; frequency 9.22 GHz. Spectra recorded on a JEOL ME-IX spectrometer. the nitrogen content of its environment and in nonphotosynthetic heterotroph, Spirillum lipo- this way to closely control the amount of energy ferum, (19) suggesting that this organism may consumed by its N2ase system. also regulate its N2ase activity by the intercon- The data presented here indicate that the versions of N2ase A and R under appropriate requirement for Mn2+ for growth of R. rubrum environmental conditions. The fact that the and R. capsulata on N2 (Fig. 1 and 2) can best N2ase from C. vinosum was fully active and be explained by a role in the regulation (i.e., would not respond to activating factor and Mn2" activation) of N2ase R which is the predominant (Fig. 5C) and earlier reports of high levels of form of the enzyme found in N2-fixing cells (5, Mn2+-independent N2ase activity in extracts of 24). This conclusion was reached after extensive the green-sulfur bacterium "Chloropseudom- efforts failed to show an Mn2+ requirement for onas ethylica" (8), a syntrophic culture of Chlo- either N2ase or glutamine synthetase biosyn- robium limicola and a nonphotosynthetic spe- thesis. Furthermore, the metal ion-activated glu- cies (11) suggests that among the photosynthetic tamine synthetases from these organisms use bacteria this nitrogenase regulatory system may Mg2+ and not Mn2" for activation (Fig. 4). The be restricted to the family Rhodospirillaceae. finding of Mn2' as a bound constituent of the chromatophore membrane (Fig. 7) along with ACKNOWLEDGMENTIS the activating factor, and the fact that it is This investigation was supported by a grant from the Research and Productive Scholarship Committee of the Uni- essentially removed by treatments that remove versity of South Carolina and by a U.S. Public Health Service the activating factor, further support the prop- grant AI-16040-01 from the National Institute of Allergy and osition that the N2ase activation system of Lud- Infectious Diseases. den and Burris (17) plays an important physio- I thank Robert Carithers for his critical reading of the manuscript and Sheryl Rhoden of the South Carolina Occu- logical role in the regulation of R. rubrum N2ase pational Health Laboratory at Columbia for the manganese activity. analysis by atomic absorption spectroscopy. 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